Abstract: Canine babesiosis is emerging vector-borne disease of dogs and wild canids. The study aimed to develop PCR assay for detection of Babesia gibsoni and understanding epidemiology of disease in dogs. The cytochrome b gene base PCR assay was developed with analytical sensitivity of detection up to 10-2 (0.5 ng/µl) of DNA. The specificity was confirmed by testing DNA from B. vogeli, Ehrlichia canis, Hepatozoon canis, and Dirofilaria repens. The thin blood smears examination revealed prevalence of 9.75% (32/328) for B. gibsoni. Whereas, with PCR the prevalence rate of 10.97% was recorded. There was significant association between vomition and dark yellow coloured urine with B. gibsoni infection. While age, breed, sex of the host and presence of ticks on animal body were not significantly associated with the occurrence of B. gibsoni infection. The sequence analysis showed 99.16-99.63% identity among themselves and 98.41 to 98.69% similarities with the published sequences of B. gibsoni. The phylogenetic analysis of Indian B. gibsoni isolates formed a single major group with other Asian countries indicating the monophyletic nature of B. gibsoni compared to other Babesia spp. Even though the vomition and yellow coloured urine combined with microscopic examination may help in diagnosis of B. gibsoni infection. Considering high analytical sensitivity and specificity of the cytochrome b based PCR, we recommend confirmatory diagnosis with PCR before undertaking the treatment in suspected cases of B. gibsoni infection.