Successful malaria management in Mali includes the use of pyrethroids and insecticide-treated nets (ITNs) for mosquito control; however, management is threatened by the spread of insecticide resistance detected via the knockdown resistance (kdr) allele. In a preliminary study, we compared the knockdown times of Anopheles gambiae from Mali using a novel ITN bioassay and the World Health Organization (WHO) bottle bioassay. Additionally, the frequency and relationship between kdr genotypes, molecular forms, and pyrethroid resistance were analyzed. The S molecular form was predominant and accounted for 76% of the assayed population. Both kdr resistant alleles, West Africa resistant (kdr-w) and East Africa resistant (kdr-e), were observed. There was no significant difference in knockdown time based on kdr genotype or molecular form of individual mosquitoes, but mosquitoes in the ITN bioassay homozygous for the kdr-w allele were knocked down significantly faster than those in the WHO bottle bioassay. The ITN bioassay provides an additional indicator of insecticide efficacy because ITNs, frequently used within homes, are the most common form of vector control and malaria prevention, and the ITN bioassays can evaluate seasonal field effects.