Blastocystis is one of the most common intestinal parasites in humans worldwide. To understand its prevalence and to explore the risk factors for Blastocystis in humans in developing countries, a molecular epidemiological investigation of Blastocystis was conducted in ethnic minority groups on both sides of the China-Myanmar border. A total of 461 fecal specimens were collected from 289 Yao people in China and 172 Wa people in Myanmar, together with a questionnaire for each participant. Based on sequence analysis of the partial small subunit of ribosomal RNA (SSU rRNA) gene (barcode region or 260 bp region), an average prevalence of 6.29% (29/461) was observed, with 4.50% (13/289) in Yao people and 9.30% (16/172) in Wa people. Twenty-two Blastocystis isolates were successfully subtyped by sequence analysis of the barcode region. Three subtypes were identified: ST1 (n = 7), ST3 (n = 13) and ST4 (n = 2). A statistical difference in the prevalence of Blastocystis was only observed between children (12.37%, 12/97) and adults (4.95%, 16/323), and between not washing hands (11.02%, 14/127) and washing hands (4.76%, 15/315) after using toilets, emphasizing the importance and necessity of health education in people in the investigated areas, especially in children.