Rye offers potential to increase the genetic variability and introduce desirable characters for wheat improvement. Triticale x wheat hybridization is a conventional method with the potential for the introduction of desirable genes from rye into wheat and has been continuously attempted by several researchers. Cytogenetic technologies now enable wheat breeders to screen lines containing rye chromatin. But these techniques are laborious. The development of molecular markers has opened up numerous possibilities for their application in plant breeding. In the present study, DNA samples from wheat, rye and triticale were amplified with inter-simple sequence repeat (ISSR) and random primers. Rye genome – specific ISSR and random amplified polymorphic DNA (RAPD) products were identified, cloned, sequenced and converted to SCAR markers. These markers were used to screen advanced generations of triticale x wheat lines from breeding programs for the presence of rye chromatin. Chromosomal locations of these markers were determined using fluorescence in situ hybridization (FISH).