We investigated the occurrence of the L1014F and L1014S kdr mutations in malaria vector populations in Burkina Faso (West Africa). A cross-sectional survey was conducted at 10 sites all located in cotton cultivation areas which are assumed to be the major insecticide resistance selection foci in Burkina Faso. The hot ligation method was used to detect the two kdr mutations in field collected Anopheles gambiae s.l. samples. For the first time in Burkina Faso the L1014S mutation was identified in both M and S forms of An. gambiae s.s. populations collected from the site of Koupela in the central-eastern region at low frequency. Furthermore, the L1014S mutation was also found in one specimen of An. arabiensis collected from the Dano site. The data generated in this study provides additional evidence of the spread of the L1014S mutation into An. gambiae s.l. populations in West Africa. It is now important to evaluate the role of the L1014S mutation in the pyrethroid resistance phenotype and assess its potential impact on the efficacy of pyrethroid-based control measures in West Africa where several resistance mutations now coexist.