Anopheles subpictus Grassi is one of the most abundant malaria vector mosquitos in Indian subcontinent especially in post monsoon months. This taxon has been speculated to be composed of four sibling species recognised using morphological and cytogenetic parameters provisionally named A, B, C and D. One of the sibling species ‘B’ has well documented status as a vector of malaria parasite in the Oriental region. Molecular phylogenetic analysis of this mosquito was done to discern sibling/cryptic species using internal transcribed spacer 2 region of the nuclear ribosomal DNA (rDNA-ITS2). An. culicifacies sibling species A, a member of Myzomia series, was used as an out-group to root the trees. In the present analysis rDNA-ITS2 region was PCR amplified, sequenced and the sequences obtained were then subjected to phylogenetic analysis with the sequences already present in the sequence database GenBank. Multiple sequence alignment was performed using ClustalX and further manually annotated in MEGA 4. Initial analysis suggested extreme 3′ end sequence divergence within different populations. Phylogenetic analysis of this spacer was done using maximum parsimony, maximum likelihood, and distance matrix—neighbour joining methods performed with bootstrapped dataset that were executed in DNAPARS, DNAML, DNADIST and neighbour programs in Phylip software package respectively. Phylogenetic trees produced were similar in topology but differed in bootstrap support. Results support the division of this taxon into at least two major clades differing in their sequence composition and product size that are recommended to be renamed as An. subpictus inland form and An. subpictus coastal form. Differences include insertions/deletions, transitions and transversions; especially one large 3′ end deletion event. The sibling species status of An. subpictus has been analysed critically.