Cryptosporidium parvum is a zoonotic pathogen composed of genetically distinct but morphologically identical genotypes. Recent molecular study indicates that dogs may transmit the cattle genotype, which is known to be pathogenic to humans. Although large-scale studies of Cryptosporidium infection in dogs have been performed in several countries, the isolates were not accurately identified because of the lack of a method for molecular analysis. It is important to identify the isolates harbored in dogs, which come in close contact with humans, in order to control human cryptosporidiosis. The aim of the present study was to calculate the prevalence of Cryptosporidium infection in dogs in Osaka city, Japan, and to characterize the isolates molecularly. The prevalence was determined to be 9.3% (13/140) by PCR. All isolates were found to be Cryptosporidium canis (previously known as the dog genotype), which is thought to be non-pathogenic in humans, based on the sequencing of diagnostic fragments. These results indicate that PCR-based diagnostic methods are a useful tool for the diagnosis and molecular epidemiology of Cryptosporidium infection in dogs, and that dogs living in Osaka are not a significant reservoir for human cryptosporidiosis. It is unclear why C. canis is dominant in dogs. Further study is required to understand this partial parasitism.