Understanding chromosome structure and genome organization of Culicine mosquitoes can potentially contribute to the development of novel approaches to vector control. However, because of highly repetitive nature of the Aedes and Culex genomes, the structure of their polytene chromosomes is damaged by ectopic contacts that make the analysis difficult. Mitotic chromosomes from imaginal discs of 4th instar larvae of Aedes aegypti were tested as a source for the physical genome mapping for this mosquito. Chromosomes in imaginal discs are 10 times more abundant than chromosomes in nervous ganglia, and they do not accumulate chromosomal mutation as cell line chromosomes do. Prometaphase chromosomes in imaginal discs of Ae. aegypti are 4-5 times longer than metaphase chromosomes and can provide higher resolution for physical mapping. Cold temperature (+16°C) was proven to increase the number of the chromosomes. Hypotonic solution treatment of live larvae was proven to elongate chromosomes and improve banding patterns. We differentially stained these mitotic chromosomes with Giemsa and YOYO-1 to revile the banding pattern. We applied fluorescent in situ hybridization (FISH) procedure developed for human chromosomes to Ae. aegypti chromosomes. A strain from Culex pipiens, Cx. quinquefasciatus and their hybrids from the natural population in Virginia was successfully colonized in the laboratory. This strain can be used as a reliable source for cytogenetic studies.