The 1899–1900 season was Bristol Rovers' first in the newly-professional Southern League. Up to this point they had been playing in the Western League and Birmingham & District League, and had been moving towards professionalism over the preceding years.
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- Brighton United and Cowes both started the season playing in this division, but both resigned from the league and had their records expunged.
- Thames Ironworks kept their place in the division after beating Division Two side Fulham 5–1 in a promotion/relegation test match.
- Sheppey United were relegated after losing a promotion/relegation test match 2–1 to Watford.
- Bedminster merged into Bristol City at the end of the season.
- Thames Ironworks were dissolved in June 1900, but were re-formed as West Ham United shortly afterwards, keeping their place in the league for the following season.