Marine lakes in the Palau Islands are known to harbor unique marine fauna that have remained isolated since the formation of the lakes after the Last Glacial Maximum. We analyzed mussels from marine lakes located on different islands and conducted morphological, phylogenetic and population genetic characterization to clarify their evolutionary history. The mussels were morphologically classified into three differentiated morphs: NS, ON, and MC. Their common characteristics were consistent with the Brachidontes—Hormomya complex of the Mytilidae family. Phylogenetic analysis based on the nuclear 18S ribosomal RNA gene supported the taxonomic position of the mussels among the Mytilidae. In the mitochondrial cytochrome c oxidase subunit I (COI) gene lineage, NS-and MC-morphs were highly diverged from each other; their estimated time of divergence dates back to the mid-Pleistocene. ON-morph was more closely related to MC-morph, although the shell morphologies of ON- and MC-morphs were easily distinguishable. Population genetic analysis revealed the coexistence of highly diverged haplotypes within a population of ON-morph, indicating introgression of mtDNA among the morphs. Our data suggest that morphological differentiation of marine lake mussels can occur in a relatively short period under different environmental conditions. Thus, the marine lakes provide a unique site for the study of diversification in mussels.