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Background: SINEs (Short INterspersed Elements) are homoplasy-free and co-dominant genetic markers which are considered to represent useful tools for population genetic studies, and could help clarifying the speciation processes ongoing within the major malaria vector in Africa, Anopheles gambiae s.s. Here, we report the results of the analysis of the insertion polymorphism of a nearly 200 bp-long SINE (SINE200) within genome areas of high differentiation (i.e. "speciation islands") of M and S A. gambiae molecular forms. Methods: A SINE-PCR approach was carried out on thirteen SINE200 insertions in M and S females collected along the whole range of distribution of A. gambiae s.s. in sub-Saharan Africa. Ten specimens each for Anopheles arabiensis, Anopheles melas, Anopheles quadriannulatus A and 15 M/S hybrids from laboratory crosses were also analysed. Results: Eight loci were successfully amplified and were found to be specific for A. gambiae s.s.: 5 on 2L chromosome and one on X chromosome resulted monomorphic, while two loci positioned respectively on 2R (i.e. S200 2R12D) and X (i.e. S200 X6.1) chromosomes were found to be polymorphic. S200 2R12D was homozygote for the insertion in most S-form samples, while intermediate levels of polymorphism were shown in M-form, resulting in an overall high degree of genetic differentiation between molecular forms (Fst = 0.46 p < 0.001) and within M-form (Fst = 0.46 p < 0.001). The insertion of S200 X6.1 was found to be fixed in all M- and absent in all S-specimens. This led to develop a novel easy-to-use PCR approach to straightforwardly identify A. gambiae molecular forms. This novel approach allows to overcome the constraints associated with markers on the rDNA region commonly used for M and S identification. In fact, it is based on a single copy and irreversible SINE200 insertion and, thus, is not subjected to peculiar evolutionary patterns affecting rDNA markers, e.g. incomplete homogenization of the arrays through concerted evolution and/or mixtures of M and S IGS-sequences among the arrays of single chromatids. Conclusion: The approach utilized allowed to develop new easy-to-use co-dominant markers for the analysis of genetic differentiation between M and S-forms and opens new perspectives in the study of the speciation process ongoing within A. gambiae. [ABSTRACT FROM AUTHOR]