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:
- ^ Bath City were deducted three points.
- ^ Fisher Athletic were deducted three points.
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.
- ^ Rothwell Town were deducted three points.
- ^ Burnham were deducted one point.
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.
- ^ Blakenall were deducted three points.
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