The species composition and genetic diversity of the subgenus Mus in Myanmar are not yet fully understood. In this study, mice were trapped in rural areas near the Ayeyarwady River basin, spanning five Myanmar cities from north to south: Mandalay, Bagan, Magway, Pyay, and Yangon. Mitochondrial cytochrome b (Cytb) and nuclear melanocortin 1 receptor (Mc1r) gene sequences were determined for mice sampled and revealed a widespread occurrence of Mus nitidulus in central Myanmar in addition to its previously known occurrence in the Yangon district of southern Myanmar. Analyses of Cytb revealed two haplogroups with a genetic distance of 2.0%, suggestive of divergence several hundred thousand years ago. Mus caroli and M. musculus were confined to Yangon and Mandalay/Bagan/Magway, respectively. Mice collected from a locality on the eastern side of the Ayeyarwady River in Pyay were identified using Cytb and Mc1r sequences as M. fragilicauda, which was previously identified only in Laos and Thailand. The species M. booduga and M. cervicolor previously predicted to be common in the study area were not found. These findings elucidate the species and genetic diversity of the subgenus Mus in the Indo–Malayan Region.