The cytogenetic profile of two Nearctic and one Paleartic treehole mosquitoes belonging to theCoelodiazesisgroup,Anopheles barberi, Anopheles judithae, andAnopheles plumbeus, was outlined by means of standard banding techniques andin situhybridization. Results indicate that, during the evolution of this species group, several rearrangements have occurred including inversions, translocations, and loss of heterochromatic material and rRNA genes copy number. The karyotype ofA. barberi ismarkedly different from the other two species, in spite of morphological similarities with the other North American species,A. judithae. This latter species has retained part of a more ancestral karyotype still shared with the PalearticA. plumbeus. Chromosomal repat‐terning inA. barberihas probably facilitated isolation and divergence of the two Nearctic species, impairing introgressive hybridization withA. judithae.