The northern house mosquito (Culex pipiens pipiens L.) is a vector of several important pathogens and comprises two epidemiologically distinct ecotypes (molestus Forskål and pipiens). The delimitation of its ecotypes is a crucial, yet controversial step in vector surveillance due to varying diagnostic values of different characters. Therefore, we reviewed the success of a diagnostic assay based on the mitochondrial cytochrome c oxidase subunit I locus (COI) by analyzing previously published sequences of molestus and pipiens sampled in different geographical areas. Next, by genotyping individuals from Northern Serbia at this locus, we additionally assessed whether genetic structure of urban and rural Cx. p. pipiens ecotypes corresponded to the admixture pattern. Finally, to account for the different susceptibility of genetic markers to introgression, we also analyzed genetic structuring based on the ribosomal internal transcribed spacer 2 (ITS2). No latitude-dependent differentiation of Cx. p. pipiens ecotypes was found at a global level, with the COI assay further failing to accurately identify molestus and pipiens ecotypes. Likewise, both individual- (BAPS) and population-based (analysis of molecular variance and FST estimates) methods showed no significant urban/rural genetic differentiation in Serbia, indicating unhindered gene flow between different Cx. p. pipiens habitat types. The findings challenge the previous instances of Cx. p. pipiens ecotype identification, while also spotlighting the vectorial capacity of their hybrid offspring.