On 20 January 1523, disloyal nobles forced her father to abdicate and offered the throne to his uncle, Duke Frederick of Holstein.

Three-year-old Dorothea and her sister and brother followed their exiled parents to Veere in Zeeland, the Netherlands, and were taken care of by the Dutch regents, their grandaunt and aunt, Margaret of Austria and Mary of Hungary.

As the eldest surviving child of the abdicated Christian II, Dorothea had a claim to the Danish, Norwegian and Swedish thrones.

Frederick had served as a loyal ally of the Emperor, and long desired to marry into the Imperial house.

The Habsburg family selected Frederick to be her consort, as they believed that he could successfully claim the Danish throne through marriage.

The couple had similar characters, shared a love for adventure and festivities, and she "was always ready to accompany him on perilous journeys, to climb mountains or ford rivers, with the same unquenchable courage and gaiety of heart.

In her anxiety to conceive, she went on pilgrimages and wore holy girdles: "this was done without any spirit of devotion, but with great mirth and laughter.

"[3] In 1537–38, she hosted her sister Christina, the Dowager Duchess of Milan, during her journey back to the Netherlands from Italy.

[5] Frederick and Dorothea never gave up her claim to the throne of Denmark, and worked actively to have the Emperor support it.

In February 1540, Dorothea was commissioned by Frederick to visit and plead her father's cause with the Emperor, to prevent a renewal of the truce between the Netherlands and King Christian III.

After consulting Archbishop Carondelet, the President of the council, and Granvelle, Dorothea and Christina sent the following petition to the Emperor: The appeal was unsuccessful however.

The Emperor officially acknowledged Christian III of Denmark the same year, but Frederick continued to press her claim.

There were considerations of marriage to a son of Philip of Hesse, or to Albert Alcibiades, Margrave of Brandenburg-Kulmbach, but none came to fruition, and the latter died in 1557.