Abstract Background There have been recent reports of surge in resistance to insecticides in pocketed areas in Ghana necessitating the need for information about local vector populations and their resistance to the insecticides approved by the World Health Organization (WHO). We therefore studied a population of malaria vectors from Kumasi in the Ashanti Region of Ghana and their resistance to currently used insecticides. We conducted susceptibility tests to the four major classes of insecticides by collecting larvae of anopheline mosquitoes from several communities in the region. Surviving adults from these larvae were then subjected to the WHO-approved susceptibility tests and characterization of knockdown resistance and acetylcholinesterase mutant genes. Results Out of 619 Anopheles specimens sampled, 537 (87%) were identified as Anopheles gambiae ( sensu stricto ), which was also the species with the lowest knockdown resistance mutant gene, 61% ( P = 0.017). Knockdown resistance mutant gene was as high as 91% in An. coluzzii . Mosquitoes collected showed susceptibility ranging from 98–100% to organophosphates, 38–56% to carbamates and 15–47% and 38–46% to pyrethroids and organochlorides, respectively. The knockdown resistance mutation frequency of Anopheles gambiae ( sensu lato ) mosquitoes that were exposed to both pyrethroids and organochlorides was 404 (65%). Acetylcholinesterase mutant gene was not found in this population of vectors. Conclusion Our study shows that pyrethroids have the highest level of resistance in the population of mosquito vectors studied probably due to their frequent use, especially in impregnation of insecticide-treated nets and in insecticides used to control pests on irrigated vegetable farms. We recommend studies to monitor trends in the use of all insecticides and of pyrethroids in particular.