The 2000–01 Ukrainian Second League was the tenth season of 3rd level professional football in Ukraine.
The competitions were divided into three groups according to geographical location in the country – A is western Ukraine, B is southern Ukraine and Crimea, and C is eastern Ukraine.
The groups were won respectively by FC Polissya Zhytomyr, FC Obolon Kyiv and FC Naftovyk Okhtyrka.
The following team were promoted from the 2000 Ukrainian Football Amateur League: Also, eight more clubs were admitted additionally: The following team were relegated from the 1999–2000 Ukrainian First League: