Background: Blastocystis is a ubiquitous, globally distributed intestinal protist infecting humans and a wide range of animals. Several studies have shown that Blastocystis is a potentially zoonotic parasite. A 1996 study reported a 70% Blastocystis prevalence in Brisbane pound dogs while another study found that pet dogs/cats of 11 symptomatic Blastocystis infected patients harboured at least one Blastocystis subtype (ST) in common with the patient. These results raised the possibility that dogs might be natural hosts of Blastocystis. In this study, we aimed to investigate this hypothesis by estimating the prevalence of Blastocystis carriage and characterising the diversity of STs in dogs from three different environmental settings and comparing these STs with the range that humans harbour. Methods: Two hundred and forty faecal samples from dogs from three different geographical regions with varying levels of socio-economic development and sanitation, namely i) 80 pet and pound dogs from Brisbane, Australia, ii) 80 semi-domesticated dogs from Dong Village, Cambodia and iii) 80 stray dogs from the densely populated cities of Sikkim, Delhi and Mumbai in India, were screened for Blastocystis using PCR and subtyped based on the ``barcode region'' of the small subunit ribosomal RNA (SSU rRNA) gene. Results: The prevalence of Blastocystis in dogs from Brisbane and Cambodia was 2.5% (2/80) and 1.3% (1/80), respectively, in contrast to 24% (19/80) in stray dogs from India. Stray dogs in India carried a diverse range of Blastocystis STs including ST 1, 4, 5 and 6 while the dogs from Brisbane carried only ST1 and one Cambodian dog carried ST2. Conclusion: The results suggest there is geographical variation in Blastocystis prevalence and STs between dog populations as reported in human studies. In addition, the greater diversity of STs and higher prevalence of Blastocystis in Indian stray dogs compared to pet/pound and community dogs in Australia and Cambodia could reflect close proximity to humans and other animals and exposure to their faeces. It appears that dogs are not natural hosts for Blastocystis but rather are transiently and opportunistically infected with a diversity of STs.