The mutant allele frequency of the Pyruvate kinase (PK) gene has been investigated mostly in pure breed cats. We investigated the PK mutant gene in stray and animal hoarding mongrel cats in Hokkaido, Japan. We also investigated the kinship of individuals carrying the mutant gene. Genotyping was conducted using the previously reported real-time PCR method. Fourteen microsatellite markers were used to identify the parents and offspring of cats carrying the PK mutant gene, and some kinship such as parent-offspring and siblings was observed. Some stray and animal hoarding cats carried the PK mutation gene and that consanguinity was confirmed among these cats indicated that the PK mutation gene was spread by unregulated interbreeding.