Blastocystis is an enteric protozoan parasite commonly found in humans and animals. Phylogenetic and genotypic analyses have shown that Blastocystis exhibits extreme genetic diversity, and humans are host to a number of zoonotic isolates. In the present study, the prevalence of Blastocystis in 276 stool samples from a hospital in Singapore was examined, and for the first time, riboprinting using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to determine the genetic diversity of the Blastocystis isolated from the Singapore population. The prevalence rate was determined to be 3.3% (9/276), and Blastocystis displaying two main ribotypes were isolated. As a comparison, we performed PCR-RFLP using two different published methodologies, and both methods allowed the isolates to be divided into two distinct groups based on their riboprint patterns. According to a recently proposed classification scheme, 78% (7/9) of the isolates were of subtype 3, while 22% (2/9) were subtype 1. The predominance of subtype 3 in an urbanized city state such as Singapore is in agreement with the idea that subtype 3 is a genotype of human origin.