Anopheline mosquitoes are important vectors of human malaria. Next-generation sequencing opens new opportunities for studies of mosquito genomes to uncover the genetic basis of a <i>Plasmodium</i> transmission. Physical mapping of genome sequences to polytene chromosomes significantly improves reference assemblies. High-resolution cytogenetic maps are essential for anchoring genome sequences to chromosomes as well as for studying breakpoints of chromosome rearrangements and chromatin protein localization. Here we describe a detailed pipeline for the development of high-resolution cytogenetic maps using polytene chromosomes of malaria mosquitoes. We apply this workflow to the refinement of the cytogenetic map developed for <i>Anopheles beklemishevi.</i>Published version