The high objectives to this study were to determine and evaluate incidence of Sarcocystis fusiform infection in slaughtered buffaloes in Assiut abattoir, and additionally study the effect of chilling on their viability and infectivity. Tissue specimens were collected from three hundred and fifty-six buffaloes (337 male buffaloes and 19 female buffaloes) slaughtered at Assiut abattoir during the period from 2/2022 to 1/2023. Samples including esophagus, tongue, and masseter muscles were examined macroscopically and histopathologically. The total prevalence of macroscopic Sarcocystis (S. fusiform) in examined buffaloes was 8.43%, the infection rate in males (Less than 2 years) and in females buffaloes (4 to 7 years old) were (8.3%) and (10.53%), respectively. The predilection seats of S. fusiform in examined buffaloes were; esophagus (90%), followed by tongue (23.3%), throat muscles (20%) and the lowest one skeletal muscle (total body) (3.3%). Concerning seasonal variation the high infection rate was happened and detected in spring (13.9%) while the lowest infection was happened in the autumn season (5.4%). The effect of chilling on the viability of S. fusiform cysts was investigated using a vital stain (0.4% trypan blue). Heavily infected muscles were chilled at 4°C for 24, 48 and 72h and was carried out on cats. The reduction rate of S. fusiformis bradyzoites after chilling was (0%), (20%) and (54.6%), respectively. However, the parasite lost its infectivity after chilling for three days, where heavily infected muscles cooling at 4°C were rendered non-infective to cats. Conclusion: The present study concluded that S. fusiformis infection in buffaloes constitutes one of the main causes of economic losses in Assiut slaughterhouses. The incidence of S. fusiform was higher in aged female buffaloes and esophagus was the main predilection seat. Additionally, our results showed that cooling heavily infected muscles with S. fusiformis at 4 °C leads to a significant reduction that touched as a fact in parasite viability and trypan blue stain effectively measured the viability of Sarcocystis.  Also, the infectivity of the parasite to cats was lost after chilling for three days.