Simple SummaryStick and leaf insects are herbivorous species widely distributed in tropical and subtropical areas, disguising themselves as leaves, twigs, or moss through morphology and behavior to avoid visually hunting predators. Currently, Phasmatodea present difficulties in taxonomy, and their phylogeny is unresolved. Mitochondria, as maternally inherited organelles, also contain evolutionary information. Compared to nuclear genes, mitogenomes have become a powerful marker for inferring phylogenetic relationships due to advantages including fast evolution rates, conserved structure, and easy amplification. With rapid advances in sequencing technology and assembly algorithms, mitogenomes can be sequenced in a very cost-effective way. As of March 2023, there are thirty-seven complete or nearly complete Phasmatodea mitogenomes listed in the NCBI. Considering the richness of Phasmatodea, additional study is warranted. In the present study, nine new mitogenomes were sequenced to examine gene rearrangements and phylogenetic relationships within the Phasmatodea.AbstractThe classification of stick and leaf insects (Order Phasmatodea) is flawed at various taxonomic ranks due to a lack of robust phylogenetic relationships and convergent morphological characteristics. In this study, we sequenced nine new mitogenomes that ranged from 15,011 bp to 17,761 bp in length. In the mitogenome ofCarausissp., we found a translocation oftrnRandtrnA, which can be explained by the tandem duplication/random loss (TDRL) model. In theStheneboea repudiosaBrunner von Wattenwyl, 1907, a novel mitochondrial structure of 12S rRNA-CR1-trnI-CR2-trnQ-trnMwas found for the first time in Phasmatodea. Due to the low homology of CR1 and CR2, we hypothesized thattrnIwas inverted through recombination and then translocated into the middle of the control region. Control region repeats were frequently detected in the newly sequenced mitogenomes. To explore phylogenetic relationships in Phasmatodea, mtPCGs from 56 Phasmatodean species (composed of 9 stick insects from this study, 31 GenBank data, and 16 data derived from transcriptome splicing) were used for Bayesian inference (BI), and maximum likelihood (ML) analyses. Both analyses supported the monophyly of Lonchodinae and Necrosciinae, but Lonchodidae was polyphyletic. Phasmatidae was monophyletic, and Clitumninae was paraphyletic. Phyllidae was located at the base of Neophasmatodea and formed a sister group with the remaining Neophasmatodea. Bacillidae and Pseudophasmatidae were recovered as a sister group. Heteroptergidae was monophyletic, and the Heteropteryginae sister to the clade (Obriminae + Dataminae) was supported by BI analysis and ML analysis.