In situ hybridization is particularly appropriate for mapping specific DNA sequences on polytene chromosomes of Drosophila and other dipterans. This technique is based on the recognition and binding of one labeled sequence (the probe) to homologous sequences on chromosomes fixed on a microscope slide. The probes are labeled with biotin or other nonradioactive products, and the probe signal can be detected as a thin line on the chromosomes, following the shape of the classical Giemsa-stained chromosome bands, thus allowing the detection of TE insertions within the range of 50 to 200 kb. In our laboratory we work on many individuals from natural populations, and as a result we process high numbers of slides hybridized with various DNA probes of transposable elements every day. Therefore, the in situ hybridization technique we use is a simplification of earlier published protocols. This chapter presents our simplified standard in situ hybridization protocol for labeling polytene chromosomes of Drosophila with biotin and a fluorescence stain (FISH).