One of the most commonly occurring foodborne, tissue cyst-forming protozoan zoonotic parasites with public health and veterinary relevance is Sarcocystis species. This research was conducted using traditional and molecular techniques to identify the incidence of Sarcocystis spp. in 750 slaughtered bovine animals of varying age and sex (375 cattle and 375 buffalo) at various abattoirs in Aswan Governorate, Egypt. The overall occurrence of macroscopic lesions of Sarcocystis spp. in cattle and buffaloes was 20.5% and 38.4%, respectively, while the occurrence of microscopic infection was 30.4% and 70.1%, respectively. Furthermore, the disease strongly correlated with the measured variables, such as the animal's age and sex. Animals older than 5 years had the highest infection rate, and females had higher infection rates than males. The esophagus, tongue, and diaphragm had the highest rates of sarcocyst compared to other organs. Histopathology studies of sarcocyst in various tissues revealed encased, circular to elongated, basophilic sarcocysts with numerous bradyzoites embedded in muscle fibers. PCR-RFLP reports that S. cruzi, S. hominis, S. hirsuta, S. buffalonis, and S. fusiformis were the species most often found. Moreover, such frequency highlights the pressing need for an effective disease control strategy and a systematic surveillance system among bovine populations. We strongly encourage the One Health approach to reduce zoonotic spread to humans and financial losses in the livestock industry.