Meat infection with the coccidian protozoan zoonotic Sarcocystis spp. causes public health hazards and high economic loss. The current study aimed to investigate molecular identification, cyst histological examination and seroprevalence of Sarcocystis spp. in water buffaloes slaughtered in the main abattoirs of 6 Egyptian Governorates; Cairo, Giza, Beni-suef, Al-Sharqia, Qalyubia, and El-Beheira. Each buffalo was visually inspected for the presence of Sarcocystis macrocysts and blood samples were collected. Out of 900 examined water buffaloes (Bubalus bubalis), 246 (27.3%) were found to be infected based on macroscopic examination. Histological examination of the macrocyst revealed metrocytes close to the cyst's edge and elongated curving basophilic bradyzoites occupying most of the cyst. Depending on age, we categorized naturally infected buffaloes into 3 groups: young (< 2 y.), adult (2-5 y.) and old (5-10 y.). The highest infection rate was observed in older buffaloes aged 5-10 years, meanwhile female animals exhibited a higher prevalence of infection compared to males. The esophagus had the highest presence of Sarcocystis compared to other organs. Using PCR based on 18S rRNA gene and sequencing, we isolated 3 Sarcocystis species from infected tissues: S. buffalonis, S. fusiformis and S. hirsuta-like. The prepared whole cyst antigen of S. fusiformis was characterized by 9 major polypeptides (140, 120, 78, 66, 53, 39, 32, 24 and 19 kDa) on 10% SDS-PAGE. Using western blotting, we identified 2 common immunogenic reactive bands (66 and 32 kDa) against naturally infected buffalo sera and hyperimmune rat sera. Additionally, sarcocystosis positive seroprevalence rate was 46.4% (418/900) with a sensitivity of 97.6% and specificity of 100% using indirect ELISA based on S. fusiformis whole cyst antigen. In conclusion, this study highlights the molecular identification, cyst histological examination and seroprevalence of Sarcocystis sp. among buffaloes in Egypt. ELISA using the S. fusiformis whole cyst antigen could be adapted to detect antibodies to Sarcocystis sp., in live animals, with an acceptable specificity and sensitivity.