The 2000–01 season was the 86th season of the Isthmian League, which is an English football competition featuring semi-professional and amateur clubs from London, East and South East England. The league consisted of four divisions.
The Premier Division consisted of 22 clubs, including 18 clubs from the previous season and four new clubs:
Farnborough Town won the division and returned to the Football Conference after two seasons spent in Isthmian League. Slough Town, Carshalton Athletic and Dulwich Hamlet finished bottom of the table and relegated to Division One. Three fixtures left unfulfilled due to bad weather conditions.
- ^ Unfulfilled fixtures: Aldershot Town v Billericay Town, Hendon v Harrow Borough and Sutton United v Hendon.
Division One consisted of 22 clubs, including 17 clubs from the previous season and five new clubs: Three clubs relegated from the Premier Division:
Two clubs promoted from Division Two:
Initially, champions of Division Two Hemel Hempstead Town were to be promoted to Division One, but later they were refused due to ground grading. Leatherhead finished in the relegation zone were reprieved.
Boreham Wood won the division and returned to the Premier Division at the first attempt. Bedford Town and Braintree Town also get a promotion. Leatherhead finished in the relegation zone the second time in a row and were relegated along with Romford and Barton Rovers.
Division Two consisted of 22 clubs, including 17 clubs from the previous season and five new teams:
Two clubs relegated from Division One:
Three clubs promoted from Division Three:
Initially, previous season champions of Division Two Hemel Hempstead Town were to be promoted to Division One, but later they were refused due to ground grading and stayed in the division.
Tooting & Mitcham United won the division and were promoted to Division One along with Windsor & Eton and Barking. Later, Barking ceased to exist when they merged with East Ham United (11° Essex Senior League) to form Barking & East Ham United, taken place of Barking in Division One.
- ^ At the end of the season Barking ceased to exist when they merged with East Ham United to form Barking & East Ham United.
- ^ Wokingham Town deducted one point.
Division Three consisted of 22 clubs, including 17 clubs from the previous season and five new teams:
Arlesey Town and Ashford Town both debuted in the league and achieved a promotion along with Lewes. Two fixtures left unfulfilled due to bad weather conditions.
- ^ Unfulfilled fixtures: Croydon Athletic v Bracknell Town and Lewes v Hertford Town.
- ^ Abingdon Town deducted three points.
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