[9][11] The Charter stipulated that if the National Library of Wales should be removed from Aberystwyth then the manuscripts donated by Sir John Williams will become the property of the University College.

Initially, however, the Library could only claim material deemed to be of Welsh and Celtic interest without any restrictions on expensive or limited edition publications.

[24] During the Second World War, many of Britain's most valuable artworks and manuscripts were stored in the National Library of Wales, which provided the evacuated treasures with a refuge from enemy bombing raids.

[10][25][26][27] The architect Charles Holden was instructed to design a tunnel for this purpose in the outcrop of rock close to the main building, with the British Museum sharing in the costs that this incurred.

[10] The Library also received irreplaceable items from other prestigious institutions such as the Ashmolean Museum, Oxford, Dulwich College and the Royal Society.

[10] Their senior member of staff was Deputy Keeper of Printed Books, Victor Scholderer, who responded to a letter from the Director, Sir John Forsdyke, by insisting that he and his colleagues would continue to sleep in the Library so that the tunnel could be checked during the night to ensure that the air conditioning was functioning properly.

[10][29] Several other institutions donated funds to the Library as an expression of their gratitude[29] and Mrs. David Sassoon, London presented two works by Cicero that were printed at Venice in the fifteenth century.

[30] The documents and artefacts that spent World War II in the care of the National Library include an original exemplification of Magna Carta,[25] drawings by Leonardo da Vinci,[25] paintings by Rembrandt, Rubens and Velásquez from Dulwich College,[10] letters of the kings and queens of England,[25] and autographs belonging to William Shakespeare.

[42][43] In 2010, the Peniarth Manuscript collection and The Life Story of David Lloyd George were amongst the first ten inscriptions on the UK Memory of the World Register, a UNESCO record of documentary heritage of cultural significance.

[80] The National Library's rare books include collections of incunabula, sixteenth-century European imprints, private press publications, bindings and scientific works.

[13] Thanks to the collections of printed books that were donated by Sir John Williams, J. H. Davies and Edward Humphrey Owen, the Library has particularly strong holdings of publications in the Welsh language from before 1912.

Nineteen of the first twenty-two books published in Welsh are present,[12] of which fourteen were acquired from the Shirburn Castle library with the Llanstephan Manuscripts.

[30] In 1922 the National Library of Wales purchased the collection of French medieval literary texts and early illustrated books that had been assembled by Francis William Bourdillon (1852–1921).

Bourdillon's library included twenty-three editions of the Roman de la Rose and an important group of works on the Arthurian legend.

[13] During the time that the incunabula expert, Dr. Victor Scholderer, Deputy-Keeper in the Department of Printed Books at the British Museum, spent in Aberystwyth during the Second World War, he took an interest in the National Library's small collection of fifteenth-century printed books and produced a Hand-list of incunabula that was published as a supplement to the National Library of Wales Journal.

The Library's also owns works from the sixteenth-century Antwerp press of Christophe Plantin and his son-in-law, Balthasar Moretus, who published De Symbolis Heroicis (1634) with its title-page designed by Peter Paul Rubens.

[13] However, the Library also has a complete set of the Kelmscott Press publications that Sir John Williams collected, including The Works of Geoffrey Chaucer (1896).

[85] The National Library's collection of works ascribed to Euclid contains more than 300 volumes, representing 270 editions,[81] and is considered to be an important reference point for Euclidean bibliographical studies.

[81] With the subsequent additions the collection covers all of Euclid's works, including Data, Phaenomena, Optica and Catoptrica along with numerous editions of the Elements, in many languages.

[88] These archives contain many different types of document, such as charters, estate records, correspondence, literary drafts and digital materials, which range from the medieval to contemporary periods.

[11][91] An insight into the creation of prose and poetry is provided by the letters, manuscript and typescript drafts,[11][91] notebooks, proofs and other personal papers of 20th and 21st century writers.

Significant holding from these archives include draft copies of novels: Cysgod y Cryman [The Shadow of the Sickle] by Islwyn Ffowc Elis, Y Stafell Ddirgel [The Secret Room] by Marion Eames and Cyfres Rwdlan by Angharad Tomos; Saunders Lewis's letters, and the correspondence between Rhydwen Williams and Alwyn D. Rees; the diaries of Caradog Prichard and Euros Bowen; and, manuscript copies of poetry, such as Y Mynach by Gwenallt, Y Mynydd by T. H. Parry-Williams and Cerddi'r Gaeaf by R. Williams Parry.

[91] The Screen and Sound Archive[38] contains The Life Story of David Lloyd George, a 1918 biographical film, which is thought to be the first feature-length biopic of a living politician.

[94] This extensive collection of estate and family records that was preserved at Penrice Castle in the possession of Miss Talbot of Margam contains manuscript material from the twelfth to nineteenth centuries.

[97][99] Self-portraits by modern Welsh artists are also collected and include Keith Andrew, David Jones, Charles Tunnicliffe and Kyffin Williams.

[99] There is a large collection of the work of Kyffin Williams in the Library, which includes his paintings of north Wales, sketches and watercolours of the Welsh colony in Patagonia and caricature portraits.

[107][108] They are an important source for the study of mid-nineteenth century Wales and, therefore, are the most frequently used collection of maps and one of the most consulted categories of documents in the Library.

[111] In January 2015, the Library, in partnership with Wikimedia UK, appointed a full-time Wikipedian in Residence with the aim of developing further its resources on an open licence for a worldwide audience.

[114] A valuable online tool for historical research is being produced by crowdsourcing the contributions of volunteers through the Cynefin website to transcribe the apportionment documents and link them to the digitised tithe maps.

It forms the largest body of Welsh text on the Web, and as well as allowing free access for all to scholarly articles on history, literature and science, and poems and book reviews.