The complete sequence of the mitochondrial genomes of 21 species in the Penaeidae were comprehensively analyzed, and the results showed that the length of the mitochondrial genome is 15 893 to 16 071 bp, A+T content is from 64.59% to 70.61%. The K_a/K_s analysis showed that among 13 protein-coding genes (PCGs) in the mitochondria of Penaeidae species, the K_a/K_s of atp8 gene was the highest, indicating that the atp8 gene may have subjected to relatively weak selection pressure in Penaeidae. In the analysis of the different sites, it was found that the nd5 and rrnL genes were ideal molecular markers. And the codon usage analysis showed that the encoded amino acid preferences were similar. At the same time, more systematic and comprehensive ML (Maximum likelihood) and BI (Bayesian inference) methods were adopted to construct a phylogenetic tree. The results showed that the topological structure of the phylogenetic tree constructed by these two methods was completely the same, and the species of the same genus were also classified into one category or separated into a branch. This study provides reliable molecular markers for the rapid identification of Penaeidae species, and provides a theoretical basis for analyzing the genetic diversity of Penaeidae species.