The infraorder Culicomorpha is a monophyletic group within Diptera, comprising 8 families. However, the precise higher-level phylogenetic relationships between these families remain debatable. To clarify this, we utilized whole-genome sequencing to extract universal single-copy orthologues (USCOs) from all families from 46 species, including 32 transcriptome or genome databases downloaded from the NCBI database and 14 newly sequenced genome databases. By filtering the amino acid (AA) and nucleotide (NUC) sequences of USCOs from each species and evaluating their gene attributes, 2 amino acid matrices (Matrix1-kpi and Matrix1-smart) and 1 nucleotide matrix (Matrix2) were assembled. Then, based on the concatenation method under site-homogeneous and site-heterogeneous models, and the coalescent-based method under the MSC model, the phylogenetic relationships of this group were reconstructed. Additionally, we used topology tests to examine different hypotheses. The model evaluation revealed that the heterogeneous model, which accounts for variation in evolutionary rates across different genomic regions, proved to be superior to the homogeneous model for obtaining the observed topology, strongly supporting hypothesis H1, namely ((Chironomidae, Ceratopogonidae),((((Culicidae, Chaoboridae), Corethrellidae), Dixidae),(Thaumaleidae, Simuliidae))). Overall, our phylogenomic analysis based on this comprehensive data offers novel insights into the phylogeny of Culicomorpha.