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Canine babesiosis is an important worldwide, tick-borne disease caused by hemoprotozoan parasites of the genus Babesia, but there is little information about epidemiology of infection and disease in Iran. In this cross sectional study, 284 blood samples were collected from owned outdoor tick-infested dogs. All sampled dogs were fully clinically examined and their collected blood was used for hematological evaluation and DNA extraction. During clinical examination, attached semi- or fully engorged ticks of each dog were pooled in sterile tubes for classification and DNA extraction. Sixty tick-infested dogs with concurrent anemia were selected as suspected population in this study. Polymerase change reaction (PCR) was done for dog’s blood andticks’ samples by a genus and species-specific PCR kit for detection of Babesia canis and Babesia gibsoni. Based on the results, among 60 tick-infested anemic dogs, three dogs (5%) were positive in genus specific PCR and all infected with Babesia gibsoni. All 211 collected ticks were identified as Rhipicephalus sanguineus. None of the collected DNA from ticks which infested dogs were positive at genus specific PCR. This study is the first molecular identification of B. gibsoni in dogs in Iran. Further studies must be done in different geographical parts to describe the importance and epidemiology of babesiosis in canine, ticks and human population in Iran.