Fifty two teams entered the competition All draws held at FFU headquarters (Building of Football) in Kyiv unless stated otherwise.

Originally FC Ihroservice Simferopol to the last moment was considered as a member of the 2009–10 First League, yet it was given a condition to provide financial guarantees with the suspense time on July 8, 2009 (2pm LST).

With the already ongoing Ukrainian Cup competition that was drawn on July 7, 2009 (just a day ahead), the Crimean club failed to satisfy that condition.

In this round entered 17 teams from Druha Liha and winners of the Ukrainian Amateur Cup.

^(1) Qualify as Amateur Cup Champions of Ukraine 2008[2] ^(2) Match not played due to bus accident involving Dynamo Khmelnytsky with several key players receiving serious injuries.

^(5) FC Kharkiv advances into the next round due to Ihroservice Simferopol withdrawal from the professional ranks.

^(10) Goals for Naftovyk not scored – Andriy Kikot and Oleksandr Aharin, for Metalurh (D) – Fabihno.

^(9) Volodymyr Bohdanov scored on the second attempt after his penalty shot was saved by Mykola Virkovsky.