Background and Aim : Bovine mastitis is an inflammation of mammary glands usually caused by bacterial infections. The economic losses due to this disease can be very high and may include milk reduction, treatment costs, and even elimination of affected livestock. Therefore, detection of the causative agents followed by taking proper management policies for prevention and/or reduction of the disease incidence is very important. Among different genus of bacteria, mycoplasmas are the 3rd most common causative agents of mastitis in cattle. Thus, investigation of such organisms as the causative agents of mastitis in dairy farms can lead to take appropriate antibiotic therapy in the field and prevent or at least decrease economic losses caused by these bacteria. Methods : Fifty bulk milk samples of dairy cattle were collected from 43 dairy cattle farms in Hamedan province of Iran. The samples were collected in a 6 months period at three stages and transferred to the lab in an ice box. Immediately after transferring the samples to the laboratory, they were evaluated by somatic cell count (SCC) assay and California mastitis test (CMT). Afterwards, DNA was extracted from all samples using a commercial DNA extraction kit and the quality of DNA samples were assessed by PCR using a bovine-specific primer pair. The DNA samples were then examined by PCR using Mycoplasma genus-specific primers to detect possible Mycoplasma infections. Both positive (M. synoviae) and negative (DNA free water) controls were also applied. Results : The results showed that 60% of the samples were positive by SCC and CMT assays. The samples contained higher than 300,000 cell/ml were considered as positive cases of bovine mastitis in SCC test. A score of 1 or more in CMT was also considered as a positive result. On the other hand, all of the extracted DNA samples had acceptable quality and were positive for bovine DNA by PCR assay. This PCR yielded a 315 bp DNA fragment in agarose gel electrophoresis. T
