© 2020 The Japanese Society of Veterinary Science. We analyzed the genotypes of three pregnant females and their litters to investigate the phenomenon of multiple paternity in wild raccoon dogs (Nyctereutes procyonoides) using 17 microsatellite markers. If a female has mated with only one male during estrus, then the maximum number of paternal alleles will not exceed two among littermates with the same father. The results revealed two out of three litters had three or four paternal alleles at one or five microsatellite loci. Therefore, the female had mated with more than one male during estrus. To the best of our knowledge, the present study is the first to report the possibility of multiple paternity in wild raccoon dogs.