In West Africa, Anopheles gambiae exists in discrete subpopulations known as the M and S molecular forms. Although these forms occur in sympatry, pyrethroid knock-down resistance (kdr) is strongly associated with the S molecular form. On the island of Bioko, Equatorial Guinea we found high frequencies of the kdr mutation in M form individuals (55.8%) and a complete absence of kdr in the S form. We also report the absence of the kdr allele in M and S specimens from the harbour town of Tiko in Cameroon, representing the nearest continental population to Bioko. The kdr allele had previously been reported as absent in populations of An. gambiae on Bioko. Contrary to earlier reports, sequencing of intron-1 of this sodium channel gene revealed no fixed differences between M form resistant and susceptible individuals. The mutation may have recently arisen independently in the M form on Bioko due to recent and intensive pyrethroid application.