Abstract: Objective Mitochondrial genes have proven to be highly effective and precise tools for species identification, origin tracing, and evolutionary research. Although complete mitochondrial genomes of 23 species in Portunidae have been sequenced, comparative genomic analyses of mitochondrial genome characteristics and phylogenetic relationships within this family remain limited. This study aims to investigate the mitochondrial genome features of species in Portunidae, identify molecular markers suitable for rapid species identification, and elucidate the phylogenetic relationships within the family. Methods Comparative genomics approaches were applied to analyze the complete mitochondrial genomes of 23 species in Portunidae. Polymorphic site analysis was conducted to screen molecular markers for rapid species identification. Phylogenetic trees were reconstructed using Maximum Likelihood (ML) and Bayesian Inference (BI) based on nucleotide sequences of 13 protein-coding genes (PCGs), alongside divergence time estimation. Results The mitochondrial genomes of species in Portunidae exhibited high structural conservation with no gene rearrangements and significant AT bias. Selection pressure analysis revealed that all 13 PCGs underwent purifying selection. Polymorphic site analysis identified nad5, nad4, and rrnl as ideal molecular markers for rapid species identification due to their longer sequence lengths and higher proportions of variable sites. Phylogenetic analysis demonstrated monophyly in seven genera, including Lissocarcinus and Lupocycloporus. Notably, Thalamita sima clustered more closely with Charybdis than with its nominal genus Thalamita, a result supported by genetic distance analysis. Divergence time estimation suggested that species in Portunidae originated no later than the Triassic period, with Lissocarcinus representing an ancient lineage, while most extant species diverged during the Paleogene. Conclusions The mitochondrial genomes of 23 species in Portunidae species are evolutionarily conserved. The nad5, nad4, and rrnl genes serve as effective molecular markers for rapid species identification, and the genus Thalamita exhibits non-monophyletic clustering in the phylogenetic tree. This study provides crucial theoretical foundations and datasets for rapid species identification, evolutionary studies, and divergence time estimation in Portunidae, advancing our understanding of their phylogenetic framework and evolutionary history.