Microdissection of chromosomes is an invaluable tool for physical isolation of single chromosomes, chromosome arms or chromosome bands. In the present study we have generated a fluorescence in situ hybridization (FISH) probe from the microdissected Y chromosome of the domestic horse. For the identification of the Y chromosome, we used the CBG technique. A biotinlabelled probe in the FISH technique gave a specific signal on all Y chromosomes and an additional signal in the interstitial part of the long arm of the X chromosome. This area showed an additional C band detected by the CBG method and characteristic for this chromosome. The probe obtained can therefore be used for analysis of the Y and X chromosomes and their copies in the genome, and, in the case of the X chromosome, also for gene mapping.