The Chi gene encodes the flavonoid synthesis enzyme chalcone-flavanone isomerase. The complete coding sequence of the Chi gene was isolated by PCR from four cultivars of cereal rye (Secale cereale L.). Unlike most monocot and dicot plant species, S. cereale has one, rather than three introns in the Chi gene. Screening of a panel of 63 Triticeae accessions, representing 31 species, showed two intron loss events in the Triticeae tribe. One intron loss occurred early in the evolution of the Triticeae tribe, while another intron loss was only detected in S. cerealeChi. A new rye-specific PCR marker was developed based on Chi intron loss polymorphism and was shown to be effective for analysis of a wide range of intergenera Triticeae hybrids for the presence of rye genome. In addition, precise genetic mapping of the rye Chi gene was carried out based on insertion/deletion polymorphism between parents of a rye mapping population. The Chi gene was mapped on the long arm of chromosome 5R 9.3 cM distal to the restriction fragment length polymorphism marker Xscb35 and 4.4 cM proximal to the locus 3Rt encoding another flavonoid synthesis enzyme, anthocyanidin-3-glucoside rhamnosyltransferase.