Background: Blastocystis is a group of cosmopolitan gastrointestinal parasite of humans and a wide variety of animals. These anaerobic protozoans include more than 17 specific small-subunit ribosomal RNA subtypes, of which nine are found in humans with a variable geographical distribution. Until now, no study has described the Blastocystis subtypes present in Saudi Arabia. Methods: In total, 1,262 faecal samples were collected from patients with gastrointestinal complaints and asymptomatic individuals visiting two major hospitals. All samples were analysed by F1/R1 diagnostic PCR, microscopy and culture methods. The subtypes of Blastocystis sp. isolates were determined by the sequenced- tagged site (STS)-based method. Results: One-hundred-thirty-three positive cases were detected by F1/R1 diagnostic PCR, of which 122 were also positive by the culture method and 83 by direct microscopy. The sensitivities of direct microscopy and the culture method were 62% and 92%, respectively. Subtype (ST3) was the most prevalent (80.5%), followed by ST1 (14.5%) and ST2 (5%). ST4, ST5, ST6 and ST7 were not detected in this study. ST3 infections were significantly predominant (P < 0.05) among symptomatic patients. Conclusions: To our knowledge, this study provides the first run-through information on Blastocystis sp. epidemiology in Makkah city, revealing a rather moderate prevalence of 10.5% and the presence of three subtypes, ST1, ST2, and ST3. ST3 was the most predominant, particularly among symptomatic patients.