The 2000–01 Southern Football League season was the 98th in the history of the league, an English football competition.
Margate won the Premier Division and earned promotion to the Football Conference. Clevedon Town, Dorchester Town, Fisher Athletic and Halesowen Town were relegated from the Premier Division, whilst Chelmsford City, Tiverton Town, Newport (Isle of Wight) and Hinckley United were promoted from the Eastern and Western Divisions, the former two as champions. Bromsgrove Rovers and Paget Rangers were relegated to the eighth level, whilst Baldock Town and Witney Town of the Eastern Division folded at the end of the season.
The Premier Division consisted of 22 clubs, including 17 clubs from the previous season and five new clubs:
| Competition: | Southern Football League Premier Division |
| Season: | 2000–01 |
| Winners: | Margate |
| Promoted: | Margate |
| Relegated: | Clevedon Town Dorchester Town Fisher Athletic Halesowen Town |
| Matches: | 462 |
| Total Goals: | 1308 |
| Prevseason: | 1999–2000 |
| Nextseason: | 2001–02 |
The Eastern Division consisted of 22 clubs, including 17 clubs from the previous season and five new clubs:
At the end of the season Langney Sports changed name to Eastbourne Borough.
| Competition: | Southern Football League Eastern Division |
| Season: | 2000–01 |
| Winners: | Newport (Isle of Wight) |
| Promoted: | Chelmsford City Newport (Isle of Wight) |
| Matches: | 462 |
| Total Goals: | 1438 |
| Prevseason: | 1999–2000 |
| Nextseason: | 2001–02 |
The Western Division consisted of 22 clubs, including 18 clubs from the previous season and four new clubs:
At the end of the season Blakenall merged with Bloxwich Town of the Midland Alliance to form Bloxwich United.
| Competition: | Southern Football League Western Division |
| Season: | 2000–01 |
| Winners: | Hinckley United |
| Promoted: | Hinckley United Tiverton Town |
| Relegated: | Bromsgrove Rovers Paget Rangers |
| Matches: | 462 |
| Total Goals: | 1427 |
| Prevseason: | 1999–2000 |
| Nextseason: | 2001–02 |