Blastocystis is one of the most common parasites inhabiting the intestinal tract of human and animals. Currently, human Blastocystis isolates are classified into nine subtypes (STs) based on the phylogeny of their small subunit ribosomal RNA (SSU rRNA) gene. Although its pathogenicity remains controversial, the possibility of zoonotic transmission was recognized since eight of the nine STs (except for ST9) have been reported in both humans and animals. A cross-sectional study was conducted to determine the prevalence and subtype distribution of Blastocystis isolated from humans and associated animals in an indigenous community with poor hygiene in Malaysia, where the risk of parasitic infection is high. A total of 275 stool samples were collected, subjected to DNA extraction and amplified by PCR assay. The Blastocystis-positive amplicons were then purified and sequenced. Phylogenetic tree of positive isolates, reference strains and outgroup were constructed using maximum likelihood method based on Hasegawa-Kishino- Yano+G+I model. The prevalence of Blastocystis infection among humans and domestic animals by PCR assay were 18.5% (45/243) and 6.3% (2/32), respectively. Through molecular phylogeny, 47 isolates were separated into five clusters containing isolates from both hosts. Among human isolates, ST3 (53.3%) was the predominant subtype, followed by ST1 (31.1%) and ST2 (15.6%). Chicken and cattle had lower proportions of ST6 (50%) and ST10 (50%), that were barely seen in humans. The distinct distributions of the most important STs among the host animals as well as humans examined demonstrate that there is various host-specific subtypes in the lifecycle of Blastocystis.