Background: An insecticide susceptibility study was carried out on Anopheles gambiae sensu lato vectors in 11 districts in Ghana between October 2012 and January 2013. Methods:An. gambiae s.l. larvae were collected, bred under standard conditions, and 3–5 d postemerged females were used for bioassay. Between 22 and 25 female An. gambiae s.l. fed only 10% sugar were used for testing. Exposure was for 1 h (2 h for fenitrothion). An. gambiae that were knocked down were recorded every 10 min and mortalities recorded 24 h posttest. Eleven insecticides from four chemical classes were used: organochlorines, organophosphates, carbamates, and pyrethroids. Subsamples of An. gambiae were analyzed by polymerase chain reaction for species and knockdown resistance (kdr) allele determination. Results: Malathion was effective in killing An. gambiae in seven districts, fenitrothion in three districts, and propoxur in one district. The organophosphate and carbamate insecticides were effective in killing An. gambiae compared to pyrethroids and organochlorines. Of the limited samples analyzed, An. gambiae sensu stricto (39/110), An. coluzzii (66/110), and An. arabiensis (5/110) were identified. Few kdr (11/110) susceptible mosquitoes were detected. Homozygous kdr^RR (65/110) and heterozygous kdr^RS (8/110) genotypes were identified. Conclusions: An organophosphate insecticide is considered appropriate for indoor residual spraying (IRS) in the 11 districts earmarked for the IRS program in Ghana.