Bovine venereal campylobacteriosis, caused by Campylobacter fetus subsp. venerealis (Cfv), is regarded as one of the major threats to the cattle industry around the world. Abortion and infertility are two important reproductive problems in cows infected with C. fetus subsp. venerealis. Reports on the presence of Cfv are scarce in the cattle, in Iran. Therefore, the present study was designed to examine the presence of Cfv in the reproductive tract of dairy cattle either slaughtered in Shiraz abattoir or dairy herds with a history of infertility and abortion, and further to identify and differentiate this micro-organism in dairy cattle in Fars, south of Iran. A total of 95 smegma samples from the preputial cavity and the fornix of the cervical opening were collected using scraping method from bulls (n = 34) and cows (n = 61) in addition to eight samples of commercially bull frozen semen. Smegma samples were then cultured for isolation of Cfv and then the extracted DNA was examined for the presence of Cfv using an optimized multiplex PCR assay. None of the frozen semen samples examined were positive for Cfv. However, out of 95 smegma samples, thirteen animals (12.6%) were found positive for Cfv consisting of 3 males and 10 females. In conclusion, the results of the current study clearly confirmed the presence of Cfv using PCR in the slaughtered cattle and dairy farms with a history of poor fertility and abortion in Fars, Iran.