The present study was done to assess the infection rate, histopathological and molecular characterization of Sarcocystis species infecting buffaloes in Menoufia governorate, Egypt.  Different muscles of 3513 slaughtered buffaloes in three abattoirs from Menoufia governorate were inspected by naked eye. Histopathological examination was applied on infected muscles with cysts. The DNA from each individual sarcocysts was extracted, amplified and sequenced. The results indicated that the total infection rate of sarcocystosis in examined buffaloes was (12.87 %). The prevalence in females was higher (20.23%) than males was (10.42%). The most affected muscles with Sarcocystis spp. cysts were esophagus. There was slight variation in the prevalence of sarcocystosis between the three abattoirs. The occurrence of sarcocystosis was slightly higher in winter and spring seasons than Autumn and Summer seasons. The amplified DNA was yielded the expected band size. The sequence of Sarcocystis cyst in this study was aligned with similar sequences of Sarcocystis spp. which was deposited in gene bank. The sequence of 18S rRNA was highly identical (94-99%) to sequences of Sarcocystis fusiformis from Egypt and other countries. The phylogenetic tree revealed that Sarcocystis in the current study was founded in the same genetic clade with the gene bank sequences of Sarcocystis fusiformis. In conclusion, the infection rate of sarcocystosis was low in current study compared with previous studies may be due to the enhanced or proper disposal of carnivores and human feces.