Due to their high solubility and stability, neonicotinoid insecticides are able to accumulate in water bodies, affecting aquatic organisms. The aims of this study were to evaluate resistance (LC₅₀) of Anopheles messeae s.l. (Anopheles messeae and An. daciae) to the neonicotinoid imidacloprid and to search for genetic markers associated with insecticide resistance. The LC₅₀ values of these species in the collections during 2017 and 2018 were indistinguishable and were in the range of 0.027-0.051 mg/l. In general, the LC₅₀ values of the mosquitoes were comparable with values of other mosquito species of the Anopheles and Culex genera. Gene polymorphisms of the variations in intron lengths and the presence of restriction sites in introns that were potentially associated with the metabolism of insecticides were studied. Polymorphisms of the studied genes in the pair of closely related species considered overlapped, but allele frequencies were different. Part of the genetic variants arose due to insertions of repetitive elements of the genome. Two variants of the cytochrome P450 gene Cyp6AG1 in An. daciae were associated with increased resistance to imidacloprid. Possible side effects of selection on insecticide resistance in blood-sucking mosquitoes are discussed. Journal of Vector Ecology 45 (2): 220-232. 2020.