For more than 70 years, since Thomas Morgan defined his heredity theory using giant, “polytene” chromosomes of Drosophila melanogaster (fruit fly), the polytene chromosomes have been a central model system in cytogenetics and chromosome studies. The dramatic, differential chromatin compaction along the genome, producing the sequence specific, banding pattern, has provided researchers with the unique opportunity to map genes to these underlying chromosome landmarks. This was originally done in order to determine linear relationships among genes, - a task that was fulfilled with the genome sequencing in 2000. However, the genetic cause of the chromatin differential compaction, which has puzzled generations of scientists, still remains unknown. To determine the key factors of the differential organization of the chromosomes, a more precise representation and categorization of the polytene cytology, as well as a means of more accurate mapping of genes to the underlying physical structure, on a large, preferably genome scale, are needed. Here we describe and troubleshoot the protocol for our new, efficient method1, which utilizing high pressure treatment of formaldehyde fixed polytene chromosomes, allows acquisition of the maximum structural and mapping information from the resulting multiple, high resolution, light microscopy images, amenable to application of the computer vision technology2,3.