Babesia species are protozoan parasites that parasitize the erythrocytes of domestic animals and humans, causing anemia in the host. The parasites cause a zoonotic disease known as babesiosis. Polymerase chain reaction (PCR) has proven to be very sensitive for detecting Babesia in blood samples of affected animals, particular in ruminants. The purpose of the current study was to determine the presence of Babesia spp. in blood samples obtained from 2 cattle, camel and sheep in Iran. In addition, the study aimed at establishing a rapid, reliable, specific and sensitive molecular tool, the polymerase chain reaction (PCR), for the detection of Babesia spp. in ruminants. Blood samples were collected from 372 ruminants (155 cattle, 95 sheep and 122 camel) kept at the Livestock Experimental Station. The animals came from randomly selected herds located in the important livestock-production regions of Iran of Isfahan and Chaharmahal va Bakhtiary during December 2012 to March 2013. PCR was used to detect Babesia spp. in the blood samples whereby an amplified band size of 428 bp was considered positive for Babesia spp. The results show that 7.10% (n= 155), 6.56% (n= 122) and 0.00% (n= 95) of the blood samples from cattle, camel and sheep, respectively, were positive for Babesia spp.. The findings from this study revealed that there were Babesia spp. in blood taken from cattle and camel. To our knowledge, this is the first report to show the presence of Babesia spp. in blood samples of Iranian ruminants in Chaharmahal Va Bakhtiari and Isfahan provinces by PCR. These findings justify the importance of control of and eradication plans for Babesia spp. infection in cattle and camel in Iran. As diagnosis of low-level infections by the parasite is important for epidemiological studies, our findings support the power of PCR test for Babesia spp. detection in blood samples and could be easily used for routine diagnosis.