To assess the genetic diversity of domestic Japanese quail (Coturnix japonica) populations, and their genetic relationships, we examined mitochondrial DNA (mtDNA) D-loop sequences and microsatellite markers for 19 Japanese quail populations. The populations included nine laboratory lines established in Japan (LWC, Quv, RWN, WE, AWE, AMRP, rb-TKP, NIES-L, and W), six meat-type quail lines reimported from Western countries (JD, JW, Estonia, NIES-Br, NIES-Fr, and NIES-Hn), one commercial population in Japan, and three wild quail populations collected from three Asian areas. The phylogenetic tree of mtDNA D-loop sequences revealed two distinct haplotype groups, Dloop-Group1 and Dloop-Group2. Dloop-Group1 included a dominant haplotype representing most of the quail populations, including wild quail. Dloop-Group2 was composed of minor haplotypes found in several laboratory lines, two meat-type lines, and a few individuals in commercial and wild quail populations. Taking the breeding histories of domestic populations into consideration, these results suggest that domestic quail populations may have derived from two sources, i.e., domestic populations established before and after World War II in Japan. A discriminant analysis of principal components and a Bayesian clustering analysis with microsatellite markers indicated that the domestic populations are clustered into four genetic groups. The two major groups were Microsat-Group1, which contained WE, and four WE-derived laboratory lines (LWC, Quv, RWN, and AWE), and Microsat-Group2 consisting of NIES-L, JD, JW, Estonia, NIES-Br, NIES-Fr, NIES-Hn, W, and commercial and wild populations. The remaining two lines (AMRP and rb-TKP) were each clustered into a separate clade. This hierarchical genetic difference between domestic quail populations is attributed to the genetic background derived from two different genetic sources-the pre-war and post-war populations-which is well supported by their breeding histories.