The aim of this study is to determine the prevalence of Bartonella and henselae species in blood samples of dogs referred to a veterinary clinic. 100 blood samples were collected. Blood samples were collected between 2021 and 2022. DNA was extracted from all blood samples. Then PCR was used to identify Bartonella genus based on 16SrRNA gene primers with fragment length (522 bp), in the next step, gltA specific primers with fragment length (130 bp) and Nested-PCR method were used to identify B. henselae species. In this study, software (AmplifiX, made in France) was used to design primers. The results showed that four samples (4%) of the blood samples based on the genus primers were positive for Bartonella infection, and also the results showed that out of the four positive samples, three samples with species-specific primers were infected with B. henselae. It was concluded that due to the difficult growth of Bartonella bacteria, PCR technique can be a suitable method for the diagnosis of this bacterium at the level of genus and species due to its high speed, accuracy and sensitivity. Although the level of contamination was not significant, due to the zoonotic nature of this bacterium, the low level of contamination can be worrying in terms of public health. It was concluded that due to the difficult growth of Bartonella bacteria, PCR technique can be a suitable method for the diagnosis of this bacterium at the level of genus and species due to its high speed, accuracy and sensitivity. Although the level of contamination was not significant, due to the zoonotic nature of this bacterium, the low level of contamination can be worrying in terms of public health.