The use of insecticides for the control of Aedes aegypti (L.) (Diptera: Culicidae) in Colombia has indirectly influenced the susceptibility status of Culex quinquefasciatus Say populations. We evaluated pyrethroid susceptibility in two populations of Cx. quinquefasciatus in the Atlantico Department of Colombia and its possible resistance mechanism (kdr mutation). Bottle bioassays were performed for permethrin, deltamethrin, and λ-cyhalothrin in female mosquitoes of Cx. quinquefasciatus.The resistance ratios (RRs) for KC₅₀ and LC₅₀ for each insecticide in the field populations examined were determined, using the Cartagena strain as the susceptible control.The L1014F kdr mutation was identified in the para gene of the voltage-gated sodium channel (vgsc), along with its allelic and genotypic frequency. Low knockdown resistance (RR_KC50) to deltamethrin was found in Puerto Colombia and Soledad populations as well as low resistance to λ-cyalothrin in this latter population. Moderate knockdown resistance to permethrin was found in both populations. At 24 h post-exposure on the other hand, there was low resistance (RR_LC50) to permethrin in Puerto Colombia and moderate resistance in Soledad. Moderate resistance to deltamethrin was found in Puerto Colombia and low resistance in Soledad. Low resistance to λ-cyhalothrin was seen in Puerto Colombia and moderate resistance in Soledad. Variability was found in the susceptibility to the pyrethroids in the populations of Cx. quinquefasciatus evaluated, and the L1014F kdr mutation is reported for the first time as a possible pyrethroid resistance mechanism in this species in Colombia.