Background: Blastocystis spp. is a unique enteric parasite commonly found in the intestines of humans and animals. In humans, prevalence up to 60% has been reported in tropical, subtropical, and developing countries. Currently 26 subtypes (STs) of Blastocystis have been described based on sequence analysis, 9 were reported in humans. Objectives: The aim of the work was to determine the different genotypes of Blastocystis spp. in symptomatic and asymptomatic individuals in Alexandria city, Egypt. Subjects and Methods: Examination of 100 stool samples was performed to detect Blastocystis collected from patients complaining of gastrointestinal (GI) disturbances and asymptomatic individuals. PCR restriction fragment length polymorphism analysis (PCR-RFLP) and sequencing of amplified products was performed for Blastocystis subtyping. Results: Out of 47 fecal samples positive only for Blastocystis spp. by microscopy, 39 patients presented with symptoms and 8 were asymptomatic participants. Blastocystis small subunit rRNA (SSU rRNA) was successfully amplified from 27 samples; 24/39 symptomatic and 3/8 asymptomatic. Among symptomatic patients, four STs were identified; ST3 was the most common (55%) followed by ST1 (20%), ST4 (15%) and ST2 (10%). While ST2 and ST4 were identified only in patients having GI symptoms, ST1 and ST3 were found in both symptomatic and asymptomatic participants. Amplicons of 7 samples from symptomatic patients were not digested after incubation with the restriction enzymes and could not be genotyped. Conclusion:Genotyping of Blastocystis spp. from fecal samples revealed the presence of four different subtypes: ST1, ST2, ST3 and ST4 with predominance of ST3. No statistically significant association could be observed between Blastocystis STs and clinical presentation of the studied subjects.RFLP analysis of Blastocystis spp.