Cytogenetic and physical maps of polytene chromosome are indispensible for specific assembly of genome sequences, functional description of chromosomal regions, and population genetic studies together with taxonomic analysis. The presence of polytene chromosomes in malaria mosquitoes serves a unique opportunity to develop high-resolution physical maps. Keeping this in mind a cytogenetic map for Anopheles stephensi ovarian polytene chromosome is prepared by using refined squash technique that increases overall band clarity. The major malaria vector Anopheles stephensi has karyotype 2n = 6. The homologous chromosome pair of the polytene chromosome complement includes 5 arms: X, 2, 3, 4 and 5.Each arm identified by specific band position and hence mapped. The physical map provides an appropriate basis for comparative genomics, which can be used for observing inversion breakpoints, duplications and also used to distinguish species complex.