The 1900–01 season was the seventh in the history of the Southern League.
Southampton were Division One champions for the fourth time in five seasons, whilst Bristol City were the only Southern League team applied for election.
At the end of the season, test matches were held between the bottom two clubs in Division One and the top two clubs in Division Two.
However, although Brentford were promoted, Grays United were not after refusing to play extra-time in their match against Watford.
[1] Only one Southern League club, Bristol City, applied for election to Division Two of the Football League.