Canine babesiosis caused by Babesia spp. is a noteworthy tick-borne zoonotic disease of domestic dogs and wild canids. In present study, a total of 556 blood samples were randomly collected from pet dogs in eight cities of Hunan province, subtropical China. Genomic DNA was extracted and Babesia DNA was detected by amplification of partial 18S rRNA gene sequences. A total of 56 (10.1%) blood samples were tested positive for Babesia species. Sequence analysis showed that 29 dogs (5.2%) were positive for B. gibsoni, and other 27 dogs for B. vogeli (4.9%). The age and health status were considered as important risk factors for B. gibsoni and B. vogeli infections in pet dogs in this study (P<0.05). Phylogenetic analysis showed that the examined positive samples were highly clustered in the same branch with B. gibsoni and B. vogeli, respectively. This is the first molecular report of B. gibsoni infection in pet dogs in Hunan province, subtropical China. Our finding has provided a guide for the control of dog babesiosis in China and elsewhere.