There are 122 mammal species in Japan, and 37 of them are bats. Especially, the species of genus Pteropus have big body size and the ability to fly hundreds of kilometers and have important roles in pollination and seed dispersal. The Ryukyu flying fox (Pteropus dasymallus) is one of the Pteropus species in Japan, distributed in the Ryukyu archipelago, Taiwan, and possibly the Philippines, and is divided into 5 subspecies. Although they are listed as VU (vulnerable) in IUCN Red List, few genetic analyses have been conducted for their conservation. The purpose of this study is to evaluate genetic diversity and investigate the genetic structure of Yaeyama flying fox, one of the subspecies of the Ryukyu flying fox. We conducted mtDNA haplotype analysis and microsatellite analysis with the 18 markers we developed.mtDNA analysis was conducted with the samples collected in 8 islands (Miyako, Ishigaki, Kohama, Kuroshima, Hateruma, Taketomi, Iriomote, Yonaguni) We identified 39 haplotypes in 526bp of the control region of 142 samples. 14 haplotypes were shared between some islands, and haplotype network for the 8 islands did not show any clear genetic structure. However, haplotype diversity was higher in Ishigaki and Iriomote compared to other islands, and some haplotypes were only found in particular islands, so there might be some genetic structure which could not be revealed by mtDNA analysis.Therefore, we also conducted microsatellite analysis with 155 samples collected in 6 islands (Miyako, Ishigaki, Kohama, Taketomi, Iriomote, Yonaguni). As a result of genetic diversity analysis, PCoA, STRUCTURE, and calculation of Fst, Yonaguni (the west end of the distribution area) population showed clear genetic differentiation from other populations, low genetic diversity, and a high inbreeding level. Ishigaki, Kohama, Taketomi, and Iriomote (the center of the distribution area) populations had gene flow between them and high genetic diversity. Miyako (the east end of the distribution area) population showed slight genetic differentiation and had the middle level of genetic diversity. Gene flow between Ishigaki and Miyako through islands between them might be preventing inbreeding of Miyako population.We revealed genetic diversity, and genetic differentiation and gene flow between islands of Yaeyama flying fox for the first time. These results will be useful for setting