We genotyped 102 Borrelia burgdorferi sensu lato strains isolated from ticks, animals, and patients in 11 provinces in China by PCR-restriction fragment length polymorphism (PCR-RFLP) amplification of 5S (rrf)-23S (rrl) rRNA gene spacer amplicons and multilocus sequence analysis (MLSA). The results showed that Borrelia garinii was the main genotype in China (65/102) and that it was distributed mainly in northern China. Borrelia afzelii was the second most frequently found species (22/102), and it was distributed in both northern and southern China. All Borrelia valaisiana strains were isolated from Guizhou Province. Additionally, one B. burgdorferi strain was isolated from Hunan Province. Our results show the diversity and wide distribution of B. burgdorferi sensu lato in China.