Simple SummaryThe mitochondrial genome is a reliable genetic marker for reconstructing phylogeny and Pteromalidae is a diverse and complex family of chalcid wasps, but its evolutionary history is still poorly understood. In this study, we sequenced the mitochondrial genomes of four species (Muscidifurax similadanacus, M. sinesensilla, Nasonia vitripennis, and Pachycrepoideus vindemmiae) of Pteromalidae. Additionally, a phylogenetic hypothesis was reconstructed for the subfamilies of Pteromalidae that includes newly acquired mitogenomes and those deposited in NCBI. We used pairwise breakpoint distances to infer this phylogeny. Our study enriches the overall knowledge on gene rearrangement in Pteromalidae, reveals the evolutionary relationships among several major groups of Pteromalidae, accumulates molecular data for a Pteromalidae phylogeny, and provides a genetic background basis for biological control in agriculture and forestry.AbstractThe mitochondrial genomes ofMuscidifurax similadanacus,M. sinesensilla,Nasonia vitripennis, andPachycrepoideus vindemmiaewere sequenced to better understand the structural evolution of Pteromalidae mitogenomes. These newly sequenced mitogenomes all contained 37 genes. Nucleotide composition was AT-biased and the majority of the protein-coding genes exhibited a negative AT skew. All 13 protein-coding genes (PCGs) initiated with the standard start codon of ATN, excepted fornad1ofN. vitripennis, which started with TTG, and terminated with a typical stop codon TAA/TAG or an incomplete stop codon T. All transfer RNA (tRNA) genes were predicted to fold into the typical clover-leaf secondary structures, except fortrnS1, which lacks the DHU arm in all species. InP. vindemmiae,trnRandtrnQlack the DHU arm and TΨC arm, respectively. Although most genes evolved under a strong purifying selection, the Ka/Ks value of theatp8gene ofP. vindemmiaewas greater than 1, indicating putative positive selection. A novel transposition oftrnRinP. vindemmiaewas revealed, which was the first of this kind to be reported in Pteromalidae. Two kinds of datasets (PCG12 and AA) and two inference methods (maximum likelihood and Bayesian inference) were used to reconstruct a phylogenetic hypothesis for the newly sequenced mitogenomes of Pteromalidae and those deposited in GenBank. The topologies obtained recovered the monophyly of the three subfamilies included. Pachyneurinae and Pteromalinae were recovered as sister families, and both appeared sister to Sycophaginae. The pairwise breakpoint distances of mitogenome rearrangements were estimated to infer phylogeny among pteromalid species. The topology obtained was not totally congruent with those reconstructed using the ML and BI methods.