Abstract Understanding the population structure and mechanisms of taxa diversification is important for organisms responsible for the transmission of human diseases. Two vectors of West Nile virus, Culex pipiens pipiens and Cx. p. molestus , exhibit epidemiologically important behavioral and physiological differences, but the whole-genome divergence between them was unexplored. In this study, we re-sequenced and compared the whole genomes of 40 individual mosquitoes from four locations in Eurasia and North America: the Republic of Belarus, the Kyrgyz Republic, and the United States of America. Principal Component, ADMIXTURE, and neighbor joining analyses of the nuclear genomes identified two intercontinental, monophyletic clusters of Cx. p. pipiens and Cx. p. molestus. The third cluster, having a polyphyletic origin, was formed by Cx. p. pipiens and Cx. quinquefasciatus from the USA. The level of genomic differentiation between the subspecies was uniform along chromosomes. The ADMIXTURE analysis determined signatures of admixture in all Cx. p. pipens populations but not in Cx. p. molestus populations. Comparison of mitochondrial genomes among the specimens showed a paraphyletic origin of the major haplogroups between the subspecies but a monophyletic structure between the continents. Thus, our study identified that Cx. p. molestus and Cx. p. pipiens represent different evolutionary units with monophyletic origin that have undergone incipient ecological speciation.