The relationships between Borrelia species and the vector ticks, Ixodes persulcatus and Ixodes ricinus were examined in a molecular epidemiological study. We conducted a survey in the Moscow region which is a sympatric region for both species of tick. We examined 630 unfed I. ricinus and I. persulcatus, ticks collected from four different regions around Moscow within an area of 250 km 2. Eighty-four ticks were culture positive (13.3%) and the prevalence rate varied in each region from 5.7% to 42.3%. No difference was found between the total prevalence rate for both species. Eight Borrelia afzelii-like variant isolates from I. ricinus and Clethrionomys glareolus were identified as B. afzelii by flagellin gene and 16S rDNA sequence analyses. Most isolates from I. ricinus were identified as Borrelia garinii type 20047 and B. afzelii. Two isolates were identified as Borrelia burgdorferi sensu stricto (s.s.) and Borrelia valaisiana, respectively, but no B. garinii type NT29 was found. In contrast, isolates from I. persulcatus were identified as both types 20047 and NT29 of B. garinii, and B. afzelii. No B. burgdorferi s.s. isolate was found among isolates from I. persulcatus.