Blastocystis sp. is an enteric protist found in humans and a wide range of animal hosts. Genetic variations were established among the 38 different subtypes detected so far, 14 of which are commonly found in human and animal hosts. The aim of the present study is to estimate the prevalence of the common Blastocystis subtypes and evaluate the possible correlation with several variables (gender, age, symptoms, domestic animals…), among patients from the southern region of Syria. Fecal samples were collected from individuals suffering from gastrointestinal complaints. Microscopic examination along with genotype analyses using seven pairs of subtype-specific primers was performed. Our results revealed the presence of Blastocystis sp. in 46 isolates out of the 60 samples microscopically studied (76.7%); single infection was detected in 24 isolates whereas co-infection with other protozoa was identified in 22 ones. Molecular detection targeting the SSU rRNA gene revealed a 100% positive presence of Blastocystis sp. in all the samples. Genotyping results detected the presence of five different subtypes (ST1-ST5) with varying proportions. However, ST1 was the dominant subtype observed (66.7%). Mixed subtype infections were found in 9 isolates (15%). Three samples remained undefined, nonetheless. Our statistical results showed no significant correlation between Blastocystis STs infection and the different studied variables. In conclusion, this study provides the first genetic characterization of Blastocystis subtypes prevalence in patients from the southern region of Syria. ST1 distribution was highly predominant. Further molecular studies are needed to estimate the prevalence of Blastocystis sp. infection in other regions in Syria and to understand the epidemiology and sources of transmission to humans.