Anopheles stephensi is one of the most important malaria vectors in the Middle-East, the Indian subcontinent, the Far-East and is the main malaria vector in south of Iran. This vector is thought to be a single but polytypic species, despite its enormous geographical range. To examine this hypothesis, we analyzed the rDNA-ITS2 and RAPD loci in different populations of An. stephensi from Iran. rDNA-ITS2 region in all sequenced specimens of An. stephensi contained a (CA)7 microsatellite sequence. Construction of phylogenetic tree based on rDNA-ITS2 sequences revealed that there only is a minor polymorphism between the different populations, despite their vast geographical distances. RAPD-PCR could differentiate rural and urban populations of An. stephensi, but it is unclear whether these two samples represent mysorensis and the type form. Further characterization of interested RAPD fragments by cloning; have shown the nature of inverted repeats and the presence of microsatellite region (GT) in both ends near to inverted repeat sequences of primers. These results showed that An. stephensi in Iran could be considered a single species with different biological and ecological forms in different zoogeographical zones. Further studies are needed to demonstrate the relation between RAPD and microsatellite sequences and the differences seen in the field for this species. This data will serve as first report on the sequence of rDNA-ITS2 and a microsatellite-containing RAPD region, which could be used for species-specific diagnosis and differentiation of urban and rural populations in An. stephensi.