Genome biology of mosquitoes holds potential in developing knowledge-based control strategies against vectorborne diseases such as malaria, dengue, West Nile, and others. Although the genomes of three major vector mosquitoes have been sequenced, attempts to elucidate the relationship between intron and codon usage bias across species in phylogenetic contexts are limited. In this study, we investigated the relationship between intron content and codon bias of orthologous genes among three vector mosquito species. We found an antagonistic relationship between codon usage bias and the intron number of genes in each mosquito species. The pattern is further evident among the intronless and the intron-containing orthologous genes associated with either low or high codon bias among the three species. Furthermore, the covariance between codon bias and intron number has a directional component associated with the species phylogeny when compared with other nonmosquito insects. By applying a maximum likelihood–based continuous regression method, we show that codon bias and intron content of genes vary among the insects in a phylogeny-dependent manner, but with no evidence of adaptive radiation or species-specific adaptation. We discuss the functional and evolutionary significance of antagonistic relationships between intron content and codon bias.