Blastocystis sp. is an enteric unicellular, anaerobic, protist that could be isolated from humans and many other farm and wild animals, in addition to rodents, reptiles and insects. This study aimed to characterize Blastocystis isolated from fecal samples of human, cattle and sheep using restriction fragment length polymorphism (RFLP) analysis. Pre-determined samples were selected from human, cattle and sheep that showed to be positive by polymerase chain reaction (PCR) targeting the small subunit ribosomal RNA gene (SSU ‑ rRNA) then examined by RFLP analysis using HinfI enzyme to determine the molecular patterns. The results of RFLP analysis revealed the presence of three different patterns of human isolates which were in difference with the other three patterns of cattle isolates. However, sheep isolates showed only one pattern. This study shows that RFLP analysis is a simple and rapid technique that could be used to characterize and differentiate Blastocystis sp. in humans and animals.