Trichomoniasis is a venereally transmitted protozoan disease of bovines that causes vaginitis, early embryonic death and second-trimester abortion, increasing the risk of infertility. Despite its economic and health significance, it has been misdiagnosed or under-reported in Pakistan. This study aimed to describe the molecular epidemiology of Trichomonas in cattle and buffaloes on selected farms in Punjab, Pakistan. Preputial and vaginal secretions (600 samples) were collected from suspected animals across eight districts by purposive sampling, together with placentomes from aborted fetuses. Samples were cultured in InPouch TF kits, and culture-positive samples were subjected to DNA extraction and PCR. Blood samples were tested by latex agglutination. The associations of natural mating, breeding practices, infrastructure and veterinary services with infection were analysed statistically. Overall prevalence was 16.67% (100/600; 95% CI 13.9-19.9%), and differed significantly by district (chi-square = 27.60, df = 7, p = 0.0003), ranging from 0% in Burewala to 30.0% in Bahawalnagar. Natural mating was strongly associated with infection (odds ratio 12.43, 95% CI 6.32-24.46, p < 0.0001) compared with artificial insemination. These results link prevalence to local risk factors and can support control strategies; artificial insemination should be preferred over natural mating.