Analysis of banding sequences of polytene chromosomes in Palearctic (Russian) and Nearctic (North American) Chironomus entis shows strong karyotype divergence between populations on the two continents. Four out of seven chromosomal arms in the North American C. entis karyotype are characterized by sequences found only in the Nearctic. In total, 44 banding sequences are now known for this species across the Holarctic, including 22 exclusively Palearctic, 6 Holarctic, and 16 exclusively Nearctic sequences. The degree of cytogenetic differentiation between Palearctic and Nearctic C. entis populations is an order of magnitude greater than differentiation among populations within either continent, but is only one third as great as the cytogenetic distance between the sibling species C. entis and C. plumosus. C. entis is the only sibling species of C. plumosus uncovered during cytological identification of Chironomus species from more than 50 North American lakes, indicating that the plumosus sibling-species group is much smaller in the Nearctic than in the Palearctic, where a dozen sibling species are known. Cytogenetic distance values calculated between Nearctic and Palearctic representatives of both C. entis and its sibling species C. plumosus are similar, but result from different patterns of karyotype divergence. New World C. entis is distinguished from Old World populations by the 16 uniquely Nearctic sequences, four of which occur in the homozygous state. In contrast, North American C. plumosus has fewer uniquely Nearctic sequences, and only one that occurs as a homozygote. However, four chromosomal arms in C. plumosus that are polymorphic in the Palearctic show fixation, or near fixation, of Holarctic sequences in the Nearctic C. plumosus karyotype. Thus, both the fixation of Holarctic sequences, and the occurrence or fixation of distinctly Nearctic sequences, contribute significantly to karyotype divergence. Patterns of karyotype divergence in Palearctic and Nearctic populations of different Holarctic chironomid species are discussed relative to intercontinental cytogenetic differentiation in other dipterans.Key words: karyotype, inversion polymorphism, cytogenetic divergence, Chironomus.