| Tuesday, 6th October 2009 |
| | | | | | | | |
| Blue Square (Conference) Premier |
| Tamworth | (1) | 2 | York City | (1) | 3 | 1,118 | |
| Unibond (Northern) Premier |
| Durham City | (0) | 1 | Ossett Town | (1) | 2 | 98 | |
| Frickley Athletic | (0) | 1 | King's Lynn | (0) | 1 | 223 | |
| Guiseley | | P | North Ferriby United | | P | | |
| Hucknall Town | (2) | 4 | Stocksbridge Park Steels | (1) | 3 | 196 | |
| Zamaretto (Southern) Premier |
| Bedford Town | (0) | 2 | Nuneaton Town | (1) | 1 | 380 | |
| Brackley Town | (0) | 2 | Bashley | (2) | 3 | 174 | |
| Didcot Town | (0) | 0 | Banbury United | (0) | 0 | 317 | |
| Evesham United | (0) | 0 | Oxford City | (0) | 0 | 119 | |
| Truro City | (1) | 3 | Chippenham Town | (3) | 5 | 429 | |
| Unibond (Northern) Div. One North |
| AFC Fylde | (0) | 0 | Trafford | (0) | 3 | 186 | |
| Garforth Town | (0) | 1 | Chorley | (0) | 1 | 92 | |
| Ossett Albion | (0) | 1 | Lancaster City | (0) | 4 | 77 | |
| Skelmersdale United | (0) | 2 | Curzon Ashton | (0) | 2 | 195 | |
| Unibond (Northern) Div. One South |
| Brigg Town | (0) | 3 | Stamford | (0) | 2 | 112 | |
| Chasetown | (0) | 1 | Witton Albion | (0) | 0 | 410 | |
| Johnstone's Paint Trophy (Associate Members) |
| Northern Section 2nd Round |
| Accrington Stanley | | P | Shrewsbury Town | | P | | |
| Bradford City | (1) | 2 | Notts. County | (1) | 2 | 3,701 | |
| after extra time, 90 minutes 2-2, Bradford City won 3-2 on penalties | |
| Bury | (0) | 2 | Tranmere Rovers | (1) | 1 | 1,903 | |
| Carlisle United | (0) | 4 | Macclesfield Town | (1) | 2 | 1,753 | |
| Chesterfield | (0) | 3 | Huddersfield Town | (0) | 3 | 3,003 | |
| after extra time, 90 minutes 3-3, Chesterfield won 4-2 on penalties | |
| Hartlepool United | (0) | 0 | Grimsby Town | (2) | 2 | 1,675 | |
| Leeds United | (2) | 2 | Darlington | (1) | 1 | 8,429 | |
| Port Vale | (3) | 3 | Stockport County | (1) | 1 | 3,154 | |
| Southern Section 2nd Round |
| Charlton Athletic | (2) | 4 | Barnet | (1) | 1 | 4,522 | |
| Exeter City | (0) | 1 | Swindon Town | (1) | 1 | 2,006 | |
| after extra time, 90 minutes 1-1, Swindon Town won 4-3 on penalties | |
| Gillingham | (0) | 0 | Norwich City | (0) | 1 | 2,814 | |
| Hereford United | (1) | 2 | Aldershot Town | (1) | 2 | 897 | |
| after extra time, 90 minutes 2-2, Hereford United won 4-3 on penalties | |
| Leyton Orient | (0) | 1 | Brighton & Hove Albion | (0) | 0 | 1,457 | |
| Milton Keynes Dons | (2) | 2 | Southend United | (0) | 0 | 4,792 | |
| Northampton Town | (0) | 2 | Bournemouth | (1) | 1 | 1,718 | |
| Southampton | (0) | 2 | Torquay United | (2) | 2 | 9,319 | |
| after extra time, 90 minutes 2-2, Southampton won 5-3 on penalties |
