The present study is aimed to identify the prevalence of Blastocystis subtypes isolated from patients in a major hospital in northeastern Thailand. A total of 562 stool samples were examined by culture technique, and 56 Blastocystis-positive samples were analyzed further by the combination of restriction fragment length polymorphism (RFLP) followed by polymerase chain reaction with sequence-tagged site primers (PCR-STS). By RFLP profiles, Blastocystis genotypes were categorized into four groups: group A (12, 21.4%), group B (32, 57.1%), group C (10, 17.9%), and group D (2, 3.6%). By PCR-STS, only four subtypes were identified. All 12 (21.4%) isolates in group A were identified as subtype 1. Similarly, all 32 (57.1%) isolates in group B were subtype 3. In group C, 10 (17.9%) samples were all subtype 7, and two samples (3.6%) in group D were both subtype 6. Of 56 Blastocystis-positive patients, 31 (55.4%) were asymptomatic and 22 (39.4%) have gastrointestinal symptoms. No significant association was observed between the Blastocystis subtypes and the clinical features. Among the Blastocystis-positive patients, the most characteristic stool samples were loose (78.6%) and soft (17.9%). In conclusion, the most common Blastocystis spp. in northeastern Thailand was subtype 3 followed by subtype 1. Relatively minor subtypes, subtype 6 and subtype 7 which are considered as avian subtypes, were found for the first time in humans in Thailand.