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Abstract Background The primary vector of West Nile virus (WNV) in Europe, Culex pipiens , has two morphologically identical but behaviorally and genetically distinct biotypes, here referred to as Pipiens and Molestus. Pipiens and Molestus, and their hybrids, are differentially distributed across Europe and display variable patterns of blood-feeding on birds and humans across the continent, but whether host choice correlates with host preference is unclear. Methods Samples of mosquitoes were collected and subsequently biotyped using real-time PCR, following which the relative proportions of each biotype and the hybrids was recorded and their host preference analyzed using a two-choice trapping assay. Each trapping assay consisted of two BG-Sentinel type 2 traps, which were baited with CO 2 and either a synthetic human odor blend or a chicken odor blend. The trapping assays were conducted in peri-urban sites in the Netherlands, Switzerland and Greece. Results The relative proportions of Molestus and hybrids were higher in Greece than in the northern locations, while Pipiens remained the dominant biotype across all trapping locations. In Greece, the host preference of Pipiens and Molestus was for avian and human odors, respectively, whereas the host preference was reversed in the Netherlands and Switzerland. The hybrids were opportunistic in host preference regardless of trapping location. Conclusions The relative proportions of Pipiens and Molestus and their hybrids vary across Europe. The observed variance in host preference, ranging from opportunistic to weakly ornithophilic for Pipiens, from ornithophilic to mildly anthropophillic for Molestus and opportunistic for hybrids—depending on latitude—may have an impact on WNV transmission. This study highlights the discrepancy between host choice and host preference, and the efficacy of the synthetic host odor blends for surveilling the relative proportion and host preference of Cx. pipiens. This methodology provides a framework and the tools required for a more accurate assessment of vectorial capacity and prediction of WNV outbreaks, and may be used to understand the genetic mechanisms regulating host preference. Graphical Abstract