This study comprehensively analyzed the complete sequence of the mitochondrial genomes of 13 species in Palinuridae. The results showed that the length of the mitochondrial genome is 15470 to 16105 bp, and the A+T content is 62.63% to 67.11%. Analysis of the ratio of the number of non-synonymous substitutions per nonsynonymous site (Ka) to the number of synonymous substitutions per synonymous site (Ks) showed that the Ka/Ks ratio of 13 protein-coding genes (PCGs) in the mitochondria of ten Panulirus species in Palinuridae were much lower than one; this demonstrated strong purification options. In the analysis of different sites, we found that the nd5, nd4, and rrnL genes were ideal molecular markers. Codon usage analysis showed that the encoded amino acid preferences were similar. This study also adopted more systematic and comprehensive maximum likelihood (ML) and Bayesian inference (BI) methods to construct a phylogenetic tree. The results showed that the Sagmariasus first began to differentiate, and Panulirus clustered into one branch joining the Linuparus/Puerulus as sisters. The Bayesian molecular clock estimation results showed that species in Palinuridae may have originated in the Triassic, and then further differentiated into lobster species with modern characteristics. This study provides reliable molecular markers for the rapid identification of lobsters, and provides a theoretical basis to analyze the genetic diversity of lobsters.