Blastocystis hominis is an intestinal protozoan that inhabits the large intestine of humans and a wide range of animals. Blastocystis species has a worldwide distribution. The current study aimed to determine the prevalence and the genetic variety of Blastocystis sp. in Iraqi children in Salah AL-Deen province, Iraq. 150 faecal samples were collected from children (5-10 years old) who attended the Salah AL-Deen hospital during the period from March to November 2020. The results revealed that 33.3% of children (50 out of 150) were found infected with Blastocystis sp., when the polymerase chain reaction (PCR) was used. The presence of ST3 gene was at a band of 526 bp where this gene was observed in 11 samples out of 50 samples. The results also showed significant differences in the prevalence rate between rural and urban regions; between symptomatic and asymptomatic children, and between children who contacted domestic animals and those who did not contact animals (P<0.05). No significant differences in the prevalence rate were between different age groups (P>0.05). Regarding the genetic variation in subtype 3(ST3) revealed in phylogenetic tree analysis, there were three variations (transversion, deletion, and transition) which were detected through the sequence alignment, also the similarity was 97% with the sequences of Blastocystis sp. registered in GenBank. The Iraqi ST3 isolate was registered with ID: OL410286 in GenBank.