Enterocytozoon (E.) bieneusi is the most common opportunistic pathogen in humans with immunocompromised status. This microorganism has been reported in various mammals including humans and companion animals. There are at least 90 genotypes of E. bieneusi, and some of them are considered zoonotic. Although pet shops and breeding kennels are the major source of dogs for owners, only a few reports are available regarding the molecular determination of E. bieneusi in pet shop puppies, and there are no reports in breeding kennels dogs. The present study reports the molecular prevalence and genotypes of E. bieneusi in 621 pet shop puppies and 314 breeding kennel dogs in Japan. The overall prevalences of E. bieneusi infection were 6.1 and 11.8% in pet shop puppies and breeding kennel dogs, respectively. The prevalences are significantly different depending on facilities, and appointed facilities maintained high levels of E. bieneusi infection. Due to the concentrated environmental contamination and the stressful situation, which can induce an immunosuppressive status, the reinfection and/or the reactivation of microsporidiosis are likely to be the major reasons for the high levels of infection in some facilities. Regarding the sequencing data of 75 polymerase chain reaction (PCR)-positive samples, 70 isolates correspond to E. bieneusi genotype PtEb IX and the remaining five are identified as genotype CD7. The importance of pet shop puppies and breeding kennel dogs as reservoirs for E. bieneusi transmission to humans is likely to be low in Japan, since both isolated genotypes here are recognized as canine specific.