The morphology and phylogenetic of Hammondia spp, Toxoplasma gondii, Neospora caninum and Sarcocystis spp. are similar and differentiation of them need simultaneous several methods examination. The aim of this study was to detect and differentiate Hammondia spp, T. gondii, N. caninum and Sarcocystis spp infection in sheep by microscopy, PCR, serology and mouse bioassay methods. Forty head of sheep with blood samples were collected in the slaughterhouse in Mashhad area from 2014 to 2015. In this study, Sarcocystis spp merozoites were microscopically detected in 50% and 52% of tongue and masseters muscles, respectively. The results of molecular examination showed that 13 (42.5%) of brain samples were PCR positive to T. gondii. No PCR-positive reactions were found for Hammondia spp and N. caninum in muscle and brain samples. Antibody against T. gondii was only detected in one sheep (2.6%). The brain samples of PCR positive were bioassayed in the mice and T. gondii was isolated from two brain samples. In the present study, only Sarcocystis spp and T. gondii infection were detected in sheep. The absence of Neospora caninum and Hammondia spp infection in sheep indicate that this animal is not a suitable host for this infection. Keywords: Hammondia; Toxoplasma; Neospora; Sarcocystis; Sheep;

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