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| 14/09/2016 | 15:30 | SK Zápy | - | FK Mladá Boleslav | 1:2 (1:1) | |
| 20/09/2016 | 15:00 | Spartak Hulín | - | Bohemians Praha 1905 | 0:2 (0:1) | |
| 15:00 | FC Sellier & Bellot Vlašim | - | FC Hradec Králové | 0:1 (0:1) | ||
| 15:30 | FK Pardubice | - | Vysočina Jihlava | 1:3 (1:3) | ||
| 16:00 | Sigma Olomouc | - | FK MAS Táborsko | 1:2 (0:2) | ||
| 21/09/2016 | 15:00 | FK Litoměřice | - | Baník Ostrava | 2:1 (0:0) | |
| 15:30 | FC Hlučín | - | Viktoria Plzeň | 0:7 (0:2) | ||
| 15:30 | Velké Mezirící | - | FK Dukla Praha | 1:2 (0:0) | ||
| 16:00 | FK Ústí nad Labem | - | Zbrojovka Brno | 1:4 (1:1) | ||
| 27/09/2016 | 14:30 | MFK Karviná | - | 1. SK Prostějov | 2:0 (0:0, 0:0) aet | |
| 28/09/2016 | 15:00 | MFK Chrudim | - | Slavia Praha | 1:3 (0:1) | |
| 05/10/2016 | 15:00 | FK Tachov | - | FC Fastav Zlín | 0:4 (0:2) | |
| 11/10/2016 | 15:00 | FK Králův Dvůr | - | FK Jablonec | 5:3 (1:1, 1:1, 1:1) pso | |
| 17:00 | 1. FC Slovácko | - | SFC Opava | 3:4 (1:0, 2:2) aet | ||
| 12/10/2016 | 15:00 | FC Písek | - | Slovan Liberec | 1:5 (1:4) | |
| 15:00 | Dynamo České Budějovice | - | AC Sparta Praha | 1:2 (0:0) |
