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| 16.09.2009 | 18:00 | SFC Opava | - | Baník Ostrava | 3:5 d.p. | |
| 22.09.2009 | 16:00 | Tescoma Zlín | - | Slovan Liberec | 1:3 d.p. | |
| 16:30 | FK Dukla Praha | - | Viktoria Plzeň | 1:4 | ||
| 23.09.2009 | 16:30 | HFK Třebíč | - | 1. SC Znojmo | 0:1 | |
| 16:30 | FC Písek | - | FK Baumit Jablonec | 1:4 d.p. | ||
| 16:30 | Viktoria Žižkov | - | Dynamo České Budějovice | 0:1 | ||
| 16:30 | SK Slovan Varnsdorf | - | 1. FK Příbram | 0:1 | ||
| 16:30 | FC Hradec Králové | - | Bohemians Praha | 2:1 | ||
| 16:30 | Sokol Protivanov | - | Sigma Olomouc | 0:5 | ||
| 16:30 | MSK Breclav | - | SK Kladno | 0:1 | ||
| 16:30 | FC Hlučín | - | FK Teplice | 1:3 | ||
| 16:30 | FK Fotbal Třinec | - | Bohemians 1905 | 0:1 | ||
| 29.09.2009 | 16:30 | Sokol Živanice | - | FC Vítkovice | 1:0 | |
| 08.10.2009 | 16:00 | 1. FC Karlovy Vary | - | Slavia Praag | 4:5 d.p. | |
| 16:00 | MFK Karviná | - | 1. FC Brno | 5:4 d.p. | ||
| 16:00 | Náchod-Destné | - | Sparta Praag | 0:6 |
