Hydatidosis is one of the most important parasitic zoonoses and remains a public health and economic problem all over the world. The hydatid cysts were collected from slaughtered cattle and sheep in Toukh abattoir, Kaliobia governorate, Egypt. Cyst fluid was obtained from hepatic and pulmonary cysts for demonstration of protoscolices and hooklets. The prevalence of infection of hydatid cyst was 12.71% and 7.87% among examined cattle and sheep respectively, 42.66% and 38.46% had hydatid cysts in liver respectively, while the infection rate was 36% and 46.15% in the lung respectively. The rate of fertile cysts was found to be 32 (61.53%) in liver and 33(64.70%) in lung of slaughtered cattle and sheep. PCR amplification was used for identification of internal transcribed spacer gene 1 (ITS1) of fertile hydatid cysts obtained from cattle and sheep by using specific primer. The amplified DNA fragment was further analyzed by PCR mediated restriction fragment length polymorphism (PCRRFLP) using two restriction enzymes (MSP1 and RSA1). The PCR yielded similar amplified DNA band of the same molecular size marker at 1115 bp in different isolates of Hydatid. No band variation of ITS1 gene could be detected by PCR- RFLP by using two restriction enzymes. Amplification product of ITSI after digestion with MSP1 showed at 661 bp and 406 bp, while those restricted with RSA1 enzyme appeared at 745 bp and 360 bp. KEY WORDS: Cattle, Cyst, Hydatidosis, PCR, Sheep