The purpose of this study was to improve our understanding of the molecular epidemiology of human Blastocystis, focusing on 239 randomly selected individuals in a single village in Yunnan province, China. Emphasis was placed on the relative frequency of different Blastocystis subtypes and underlying risk factors. We used a cross-sectional study design, by employing a pre-tested questionnaire to obtain demographic data and behavioural risk factors, and collected faecal samples for culture and subsequent identification of Blastocystis. DNA was extracted from Blastocystis isolates and the subtypes were identified using 7 subtype-specific sequenced-tagged site (STS) primers. Overall, 78 faecal samples were Blastocystis culture-positive (32.6%, 95% confidence interval: 26.7-38.6%). The majority (n=73, 93.6%) were single infections with one of the known subtypes, whereas 2 isolates consisted of 2 concurrent subtypes. The remaining 3 isolates could not be identified with the currently known STS primers. Risk factors for a Blastocystis infection were drinking unboiled water, consumption of raw water plants and pig ownership. The consumption of raw water plants was positively associated with subtype 1 infections, and drinking unboiled water with subtype 3 infections. In conclusion, human Blastocystis was common in this village in southwest China, and different subtypes were associated with distinct transmission routes or sources of infection, and hence Blastocystis subtypes might be linked to specific environmental compartments.