| 16.09.2009 | |||
| 21:00 | Mérida UD | 1-1/p.5-4 | CF Reus Deportiu* |
| 09.09.2009 | |||
| 21:30 | CA Ceuta | 2-2/p.4-5 | Alicante CF |
| 03.09.2009 | |||
| 21:00 | CD Numancia | 2-3 | Real Murcia |
| 02.09.2009 | |||
| 22:00 | UD Las Palmas | 2-1 | Cádiz CF |
| 21:30 | Albacete Balompié | 0-1 | Hércules CF |
| 21:30 | FC Cartagena | 3-2 AET | Elche CF |
| 21:00 | AD Alcorcón | 2-0 | CF Palencia |
| 21:00 | Atlético Ciudad | 3-2 | Real Oviedo |
| 21:00 | CD Alcoyano | 2-0 | Villajoyosa |
| 21:00 | CD Puertollano | 3-0 | Zamora CF |
| 21:00 | Girona FC | 1-0 | Gimnàstic Tarragona |
| 21:00 | Levante UD | 0-0/p.3-4 | SD Huesca |
| 21:00 | Ontinyent 1931 CF | 2-1 | UB Conquense |
| 21:00 | Polideportivo Ejido | 0-0/p.8-7 | SD Ponferradina |
| 21:00 | Real Betis | 1-2 | Córdoba CF |
| 21:00 | Real Sociedad | 0-2 | Rayo Vallecano |
| 20:45 | CE Sabadell | 0-0/p.1-3 | Marbella FC |
| 20:45 | UD Salamanca | 1-1/p.4-3 | CD Castellón |
| 20:30 | Cultural Leonesa | 4-0 | Unión Estepona |
| 20:30 | UE Sant Andreu | 3-1 | UD Melilla |
| 19:30 | CD Lagun Onak | 2-1 | Cerro Reyes Badajoz |
| 01.09.2009 | |||
| 21:00 | Celta de Vigo | 2-1 | Real Unión |
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