Simple SummaryChickens have long lived with humans as companion animals for religious, ornamental, food production, or entertainment purposes. Chickens cannot fly or move long distances by themselves, and their introduction history is closely related to the history of human migration and trade. Indigenous chicken breeds with unique genetic characteristics have been established according to their uses and habitat in the world. Many ancient trade routes across the Eurasian continent played an important role in bringing various chicken breeds to each area, and Japan was one of the eastern ends of trade routes 1000–2000 years ago. In this study, molecular phylogenetic analyses suggested that Japanese indigenous chickens originated mainly from China, with some originating from Southeast Asia. Population genetic analyses revealed that most Japanese indigenous chicken breeds possess unique genetic characteristics. Furthermore, genetic assessments of Japanese indigenous chickens will provide new insights into the dispersal history and genomic evolution of domestic chickens. Moreover, investigation of indigenous chicken genetic characteristics contributes to revealing the genomic evolution of chickens and discovering candidate genetic resources for developing highly productive chicken breeds in their habitat environment.Japanese indigenous chickens have a long breeding history, possibly beginning 2000 years ago. Genetic characterization of Japanese indigenous chickens has been performed using mitochondrial D-loop region and microsatellite DNA markers. Their phylogenetic relationships with chickens worldwide and genetic variation within breeds have not yet been examined. In this study, the genetic characteristics of 38 Japanese indigenous chicken breeds were assessed by phylogenetic analyses of mitochondrial D-loop sequences compared with those of indigenous chicken breeds overseas. To evaluate the genetic relationships among Japanese indigenous chicken breeds, a STRUCTURE analysis was conducted using 27 microsatellite DNA markers. D-loop sequences of Japanese indigenous chickens were classified into five major haplogroups, A–E, among 15 haplogroups found in chickens worldwide. The haplogroup composition suggested that Japanese indigenous chickens originated mainly from China, with some originating from Southeast Asia. The STRUCTURE analyses revealed that Japanese indigenous chickens are genetically differentiated from chickens overseas; Japanese indigenous chicken breeds possess distinctive genetic characteristics, and Jidori breeds, which have been reared in various regions of Japan for a long time, are genetically close to each other. These results provide new insights into the history of chickens around Asia in addition to novel genetic data for the conservation of Japanese indigenous chickens.