Mosquito feeding behavior determines the degree of vector- host contact and may have a serious impact on the risk of pathogen transmission, including that of the West Nile virus (WNV). To measure the role of Culex mosquitoes as WNV vectors, host-seeking females were collected using animal-baited traps containing live birds (quail) or mammals (rabbits) and CO₂-baited Center for Disease Control and Prevention traps placed in several wetland areas in the Czech Republic. Culex pipiens (L.) and Culex modestus (F.) were the most frequently collected species. Although Cx. modestus did not distinguish between baits, Cx. pipiens was collected significantly more frequently in bird-baited traps. Based on mitochondrial DNA analysis of bloodmeals from engorged females collected by CO₂-baited traps situated within reed beds, a diverse group of birds were the predominant hosts (93.7%), followed by mammals (4.2%) including humans, and amphibians (2.1%). Among birds, Anseriformes were fed upon most frequently by Cx. modestus, whereas Cx. pipiens fed most frequently on Passeriformes. To measure the infection risk and confirm the distribution of mosquito species in various biotopes, transects of CO₂-baited CDC traps were operated from wetland reed beds into upland vegetated areas. Even though both Culex species occurred in all biotopes sampled and frequently dispersed hundreds of meters away from fishpond shore vegetation, the spatial distribution of Cx. modestus was significantly associated with reed beds at wetlands. The first detection of WNV (subtype RabV) in Cx. modestus in Bohemia and confirmation of WNV presence in Cx. pipiens in Moravia together with observed feeding behavior supports the presumed role of both Culex species in the avian-to-avian enzootic WNV cycle and in avian-to-mammal transmission in the Czech Republic.