Studies aimed at monitoring the spread of knockdown resistance to pyrethroids (kdr) in time and space are particularly useful for detecting barriers to gene flow among the chromosomal and molecular forms of Anopheles gambiae. We used a recently developed polymerase chain reaction assay to estimate changes in kdr frequency that occurred in several mixed-form populations from Mali, West Africa, in the past decade. We found that the kdr allele significantly increased in frequency in most populations but was still absent from the M molecular form. Importantly, within the S molecular form, kdr was detected for the first time in the Bamako chromosomal form. These results provide important insights on the patterns of spread and emergence of pyrethroid knockdown resistance in West Africa.