Gastro-intestinal parasites are prevalent across the world especially in developing countries. The prevalence is high in rural and semi-urban areas. Buxtonella (B.) sulcata is an opportunistic protozoan parasite causing gastrointestinal (GI) problems in cattle and buffaloes which serve as the reservoir host for this zoonotic pathogen. The oocysts of this parasite are resistant to chemical treatment and survive well in harsh environmental conditions like temperature, humidity etc. outside the host. In Pakistan, limited published data is available on molecular studies on prevalence of this parasite in bovines and associated risk factors. Briefly, 384 faecal samples (as determined through epidemiological sample size calculation formula) were collected from cattle and buffaloes from four towns of District Faisalabad through simple random sampling method. On a pre-designed questionnaire with closed ended questions, data on certain associated risk factors of GI protozoal infections was gathered. Parasitic oocysts were separated from faeces by using centrifugal floatation technique. DNA extraction was done from isolated oocysts of parasites followed by PCR analysis using genus specific primers (18S rRNA gene). Out of total samples collected, 78 samples were found positive for Buxtonella spp. with prevalence rate of 20.31%. The highest prevalence was observed in Lyallpur town 26.17% followed by Iqbal town 19.59%, Jinnah town 14.13% and Madina town 10.23%, respectively. The infection was found higher in buffaloes as compared to cattle in the study zone. Upon phylogenetic analysis, B. sulcata showed 90% similarity with Balantidium (B.) coli isolated from pigs (Accession No. MT191774.1) through Minimum Evolution Method by using Bootstrap test of phylogeny. In conclusion, we need appropriate preventive measures in order to prevent its zoonotic transmission.