In situ hybridization with an interspersed repeat clone from rye, pSc119, was shown to be useful for detecting rye chromosomes introduced into wheat. However, since pSc119 also shows strong hybridization to a few sites in certain wheat chromosomes, small rye chromosome segments added to wheat may be difficult to detect. In this study, detection of rye chromosomes present in triticale and triticale X wheat hybrids was accomplished with the use of a subfragment from pSc119 (pSc119.1) whose sequence is dispersed throughout the rye chromosomes and only weakly cross-hybridizes to a few telomeric and centromeric regions of wheat. The in situ hybridization conditions were optimized to readily distinguish rye chromosomes from wheat chromosomes without the need for intensive analysis of hybridization patterns. Rye chromosomes were readily detected using fluorescence in situ hybridization. Fluorescence detection provided increased sensitivity over enzymatic detection and allowed signals to be amplified with repeated use of biotinylated anti-avidin antibody and avidin-FITC. Detection of rye chromatin was further optimized by doubling the probe concentration. Finally, double exposure photography of the same cell with two different filters provided another means to further increase the contrast between rye and wheat chromosomes.