Sarcocystosis is regarded as one of the most important protozoal infections of buffaloes. This study was conducted to determine the prevalence of Sarcocystis spp. in water buffaloes (Bubalus bubalis) in slaughterhouses in Sharkia province, Egypt. In addition, effect of age and sex on the infection rates was reported. Therefore, Sarcocystis isolates were identified morphologically using macroscopic, microscopic and histopathological examinations, while molecular identification was carried out using amplification of 18S ribosomal subunit RNA (18S rRNA) by PCR and DNA sequencing. The current findings showed that the prevalence of Sarcocystis spp. in water buffaloes was 41.50%. Further, using both morphological and sequences analysis indicated that the isolates obtained from buffaloes were macroscopic S. fusiformis. This is the first morphological and genetic study of S. fusiformis in slaughtered buffaloes in Sharkia province which will provide useful information for monitoring and controlling of Sarcocystis infections.