Cereal centromeres consist of a complex organization of repetitive DNA sequences. Several repetitive DNA sequences are common amongst members of the Triticeae family, and others are unique to particular species. The organization of these repetitive elements and the abundance of other types of DNA sequences in cereal centromeres are largely unknown. In this study, we have used wheat-rye translocation lines to physically map 1BL.1RS centromeric breakpoints and molecular probes to obtain further information on the nature of other types of centromeric DNA sequences. Our results, using the rye-specific centromeric sequence, pAWRC.1, indicate that 1BL.1RS contains a small portion of the centromere from 1R of rye. Further studies used molecular markers to identify centromeric segments on wheat group-1 chromosomes. Selected RFLP markers, clustered around the centromere of wheat homoeologous group-1S chromosomes, were chosen as probes during Southern hybridization. One marker, PSR161, identified a small 1BS segment in all 1BL.1RS lines. This segment maps proximal to pAWRC.1 in 1BL.1RS and on the centromere of 1B. Sequence analysis of PSR161 showed high homology to HSP70 genes and Northern hybridization showed that this gene is constitutively expressed in leaf tissue and induced by heat shock and light stimuli. The significance of this work with respect to centromere organization and the possible significance of this HSP70 gene homologue are discussed.