The Anopheles gambiae complex of mosquitoes includes malaria vectors at different stages of speciation, whose study enables a better understanding of how adaptation to divergent environmental conditions leads to evolution of reproductive isolation. We investigated the population genetic structure of closely-related sympatric taxa that have recently been proposed as separate species (An. coluzzii and An. gambiae), sampled from diverse habitats along the Gambia River in West Africa. We characterised putatively neutral microsatellite loci as well as chromosomal inversion polymorphisms known to be associated with ecological adaptation. The results revealed strong ecologically-associated population subdivisions within both species. Microsatellite loci on chromosome-3L revealed clear differentiation between coastal and inland populations, which in An. coluzzii is reinforced by a unusual inversion polymorphism pattern, supporting the hypothesis of genetic divergence driven by adaptation to the coastal habitat. A strong reduction of gene-flow was observed between An. gambiae populations west and east of an extensively rice-cultivated region apparently colonized exclusively by An. coluzzii. Notably, this ‘intra-specific’ differentiation is higher than that observed between the two species and involves also the centromeric region of chromosome-X which has previously been considered a marker of speciation within this complex, possibly suggesting that the two populations may be at an advanced stage of differentiation triggered by human-made habitat fragmentation. These results confirm ongoing ecological speciation within these most important Afro-tropical malaria vectors and raise new questions on the possible effect of this process in malaria transmission.This article is protected by copyright. All rights reserved.