Although insecticides play a crucial role in malaria control programs, this strategy is threatened by the development of resistance in the vectors to commonly used public health insecticides. Due to their known efficacy and lower toxicity to humans and non-target organisms, pyrethroids have been used in many vector control projects. In West Africa, pyrethroid resistance in the major malaria vector, Anopheles gambiae, had been reported in several countries. It was, therefore, imperative to investigate the susceptibility of this very important malaria vector in Ghana and characterize the resistance mechanism. Larvae of Anopheles gambiae sensu lato were collected from rural and urban sites and reared to adults. Species identification was by morphological characteristics, PCR, and HhaI restriction digest. Female mosquitoes that were two to three days old were selected and exposed to World Health Organization (WHO) diagnostic doses of three pyrethroids. A susceptible laboratory strain of An. gambiae sensu stricto was used as a reference. Both survivors and dead mosquitoes from bioassays were screened for the knockdown resistance mutation. An. gambiae sensu stricto was the only sibling species of the complex present in these localities with the molecular S form being predominant (>95%). Resistance to pyrethroids up to 8.5 fold was observed, with very high kdr frequency. The relative ease in using molecular techniques has resulted in rapid detection of ostensible insecticide resistance genes in malaria vectors. However, it is even more important to complement these molecular tools with routine insecticide testing in the field, especially if the insecticides are earmarked for public health use.