The development of genomic resources and tools is an important step in designing novel approaches to genetic control of mosquitoes. Physical genome maps enhance the quality of the genome assemblies, improve gene annotation, and provide a better framework for comparative and population genomics studies in mosquitoes. In this chapter, we describe protocols for an important procedure in physical genome mapping-fluorescence in situ hybridization (FISH). We provide details on (1) dissection of salivary glands, ovaries, and imaginal discs for obtaining high-quality polytene or mitotic chromosome preparations; (2) DNA-labeling procedures and extraction of repetitive DNA fractions; and (3) approaches to FISH on polytene and mitotic chromosomes.