During this edition a coin toss was used a record three times to decide winners of the ties after play-off matches ended as draws.

Italy continued to be represented by two clubs, with Inter Milan qualifying as title holders and Bologna as Italian champions.

Alongside ever-present Real Madrid were Benfica, Rangers and Dukla Prague appearing in their sixth edition.

Lokomotiv Sofia, Chemie Leipzig, Liverpool, Reipas Lahti, Győri Vasas ETO, KR, Bologna, Aris Bonnevoie, Sliema Wanderers, DWS, Glentoran, Malmö FF and La Chaux-de-Fonds made their debut in the competition.

All participants were their respective associations champions, except for Inter Milan and Malmö FF.

The latter was leading Allsvenskan at the summer break on 1 July 1964, after 11 of 22 matches had been played, but finished second in autumn of 1964.

[2] Similar to previous edition, due to the number of participating teams, only title holders Inter Milan received a bye to the first round.

The remaining clubs would play the preliminary round in September and October, apart from one match taking place in August and one play-off in November.

For the first time in the history of European Cup teams in the preliminary round were not divided geographically into pots, which resulted in some teams covering over 4,400 kilometres for their ties (e.g. DWS and Fenerbahçe), while other had shorter trips (e.g. Dukla Prague and Górnik Zabrze – around 600 kilometres).

Dukla Prague 4–4 Górnik Zabrze on aggregate; play-off needed.

The top scorers from the 1964–65 European Cup (including preliminary round) were as follows: