Abstract
Mitochondrial genomes are powerful tools for taxonomic delimitation and species identification, yet they remain scarce for Chironomidae (Diptera). In this study, we assembled and annotated 63 new mitochondrial genomes, encompassing 63 species within 39 genera in Orthocladiinae sensu lato (including Prodiamesinae and Orthocladiinae) and Chironominae by whole-genome sequencing, marking the first report of mitochondrial genome data for the Xiaomyini. Comparative analyses revealed structural variation, including transfer RNA gene rearrangements, along with strong nucleotide composition bias, codon usage patterns, and gene-specific selection pressure differences. Distinct evolutionary dynamics were detected among protein-coding genes, ribosomal RNAs, transfer RNAs, and the control region. Heterogeneity analyses and phylogenetic analyses showed that amino acid datasets perform better for basal branch of Orthocladiinae relationships, although the resolution within non-basal branches of Orthocladiinae remains limited. By substantially increasing both the number and taxonomic breadth of mitochondrial genomes in Chironomidae, this study delivers a vital foundation for future multi-marker phylogenetic reconstruction, taxonomic revision, and rapid species identification, with direct applications to biodiversity conservation and freshwater ecosystem monitoring.