Lithophaga has significant ecological value in coral reef ecosystems. In this study, the mitochondrial genomes of Lithophaga teres and Lithophaga zitteliana were newly measured, and their applications in system evolution and Lithophaga population genetics were investigated. The mitochondrial genomes of Lithophaga teres and Lithophaga zitteliana are both double-chain ring structures, which are 35,469 bp and 38,333 bp respectively, and both are composed of 2 rRNAs, 22 tRNAs and 13 PCGs. By constructing the phylogenetic tree of Mytilidae species, the taxonomic status and phylogenetic relationship of Lithophaga in Mytilidae were identified, the classification system of Lithophaga was improved, and it was clarified that Lithophaga is a monophyletic group. Combined with the mitochondrial gene composition and arrangement characteristics, Support removing Leiosolenus out of Lithophaginae. Furthermore, selection pressure analysis was conducted on mitochondrial PCGs of Lithophaga teres and Lithophaga zitteliana. The nad2 gene was screened out as a molecular marker and combined with the cox1 gene for genetic analysis of three wild populations of Lithophaga teres in the South China Sea of China. The lengths of the cox1 and nad2 gene fragments were 748 bp and 425 bp respectively. Compared with cox1, nad2 could reflect more genetic variations and was more suitable for the population genetic analysis of Lithophaga teres. Among the three populations, the Tianhougong population has a higher genetic diversity, a longer genetic distance from the other populations, and obvious differentiation. Neutral tests and mismatch distribution analyses indicate that all three Lithophaga teres populations in the South China Sea of China have recently experienced population expansion.