The parasites that belong to the genus Blastocystis are the most common intestinal parasite detected in a wide range of hosts including humans. Although the pathogenicity of these parasites remains controversial, many studies confirmed their pathogenicity and some researchers strongly believe that the pathogenicity may be linked with specific subtypes of these parasites. The current study investigated the Blastocystis sp. subtypes recovered from individuals suffering from the irritable bowel syndrome (IBS) in comparison with those recovered from non-IBS subjects. Fresh stool samples were collected from each participant and processed during the same day of collection. Iodine wet mounts and trichrome stained smears prepared from fresh stool and sediment concentrates were microscopically examined for Blastocystis parasites. In addition, Jones's medium has been used in order to confirm the identification and also to get the genomic DNA from positive samples for PCR and sequencing. The culture was significantly more sensitive (P=0.0035) than the other identification methods, especially in IBS patients. Blastocystis was detected in 60.0% of patients with IBS and in 22.0% of non-IBS individuals and the difference between two groups was statistically significant (P=0.0001). Regarding the impact of age and gender on the prevalence of infection with Blastocystis, no significant differences were observed between IBS patients and non-IBS subjects except for the age group (10-30 years) where the non-IBS subjects were significantly more prone (P=0.0223) to the infection with this parasite than IBS patients. The abdominal pain and bloating were the leading symptoms. DNA sequencing and phylogenetic analysis of Iraqi Blastocystis isolates identified three subtypes (ST1, ST2 and ST3). Among these three subtypes, ST3 was significantly more prevalent (OR=8.5; P=0.0058) among IBS patients (60%) than non-IBS subjects (25%). In contrast, the dominance of ST1 was significantly higher (OR=7.0; P=0.0062) in the non-IBS subjects (70%) than their IBS patients counterparts (15%). As far as we know, this study is the first to deal with the genetic characterization of Blastocystis subtypes in an Iraqi population with and without IBS.