Simple SummaryCanine vector-borne diseases (CVBDs) can affect the health of domestic and wild animals, and their prevalence is increasing worldwide. As potential reservoirs of zoonotic pathogens, dogs might transfer these pathogens to humans. There is limited knowledge about the vector-borne pathogens circulating in dogs in China. To investigate the current epidemiological status and genetic characteristics ofEhrlichiaspp.,Hepatozoonspp., andMycoplasmaspp., blood samples were collected from healthy pet dogs in four regions of China. There was no evidence ofEhrlichiaspp. orMycoplasmaspp., and onlyHepatozoon caniswas detected in these dogs. High haplotype diversity and the occurrence of genetic variation were observed among theseH. canisisolates. These results will be useful for developing effective control approaches against CVBDs in companion animals.AbstractCanine vector-borne diseases are widely distributed around the world. They are transmitted by arthropods, and many seriously threaten the health of animals and humans. In China, our knowledge ofEhrlichia,Hepatozoon, andMycoplasmaspecies circulating in dogs is still poorly understood. Therefore, the aim of this study was to understand the prevalence and genetic characteristics of canineEhrlichiaspp.,Hepatozoonspp., andMycoplasmaspp. in Chongqing (southwest), Fujian (southeast), Shandong (southeast), and Hubei (central) Provinces of China. Blood samples from healthy pet dogs were processed to detectEhrlichia,Hepatozoon, andMycoplasmaDNA with PCR. Haplotype and phylogenetic analyses were performed on 18S rRNA sequences. Among 306 dogs, noEhrlichiaspp. orMycoplasmaspp. were detected, whereas oneHepatozoonsp. was detected in 10 (3.27%) of the animals. OnlyHepatozoon caniswas identified and was endemic to Chongqing (2.46%) and Hubei (8.77%). A haplotype analysis identified eight haplotypes among theH.canisisolates. A phylogenetic analysis showed that theH. canisisolates in this study clustered into four clades, together with isolates from different countries and hosts, forming a large group that was clearly separate from otherHepatozoonspecies. These findings provided new information on the epidemiological characteristics of canine vector-borne diseases in China and will be helpful in the development of efficient measures to safeguard the health and well-being of companion animals and their owners.