The aim of this work was to study the prevalence and genetic diversity of Babesia in Ixodes persulcatus ticks and small mammals from Ural and Siberia in Russia. In total, 481 small mammals and 922 questing adult I. persulcatus from North Ural (Sverdlovsk region) and West Siberia (Novosibirsk region) were examined for the presence of Babesia by nested PCR based on the 18S rRNA gene. Babesia microti of the 'Munich'-type was found in 36.2% of blood samples of the small mammals from the Sverdlovsk region and B. microti of the 'US'-type in 5.3% of the animals from the Novosibirsk region. Babesia DNA was not detected in 133 analysed I. persulcatus from the Sverdlovsk region; however, it was found in 24 of 789 ticks from the Novosibirsk region. Three distinct Babesia species were detected in I. persulcatus. B. microti 'US'-type was identified in 10 ticks, Babesia closely related to B. divergens/B. capreoli in 2 ticks, and Babesia closely related to B. venatorum (EU1) in 12 ticks. To our knowledge, this is the first detection of Babesia sensu stricto in I. persulcatus ticks and of B. microti in I. persulcatus in the Asian part of Russia.