Post-partum period has an important role in cows' breeding due to its effects on reproductive efficiency and subsequent pregnancy. Escherichia coli, Trueperella pyogenes (Arcanobacterium pyogenes), Fusobacterium necrophorum and Prevotella melaninogenicus are recognized as major pathogens associated with uterine endometrial lesions. The objective of this study was to identify these pathogens using the polymerase chain reaction (PCR) as a culture-independent sensitive method. A total of 172 cows were examined 25–35 days post-partum, and 128 cows were examined at 2 weeks later (39–49 days post-partum). Uterine discharges were collected by covered plastic infusion pipettes. The prevalence of endometritis was greater in the first examination than the second (35.5% vs. 16%). E. coli was detected in eight of the samples, T. pyogenes was detected in 13 of the samples and F. necrophorum was detected in 11 of the samples. There was no positive sample of P. melaninogenicus. Uterine contamination by T. pyogenes and F. necrophorum in the first examination was higher than the second examination. T. pyogenes affected as a tendency the prevalence of clinical endometritis in first examination. Primiparous cows showed 4.02 times higher odds of clinical endometritis compared with second-parity cows in first examination. A multiplex PCR protocol as a simple, less expensive, fast assay was introduced to identify E. coli, T. pyogenes and F. necrophorum.