Genetic mapping of centromeres has been a challenge for plant geneticists. The objective of this study was to develop a new strategy for determining the locations of centromeric regions on genetic maps by mapping centromere-associated sequences, to make it possible to define the centromeric region of each chromosome as a single Mendelian locus on the molecular linkage map. Two DNA probes containing sequences specifically associated with the centromeres of grass species were used for genetic mapping. The centromere-associated sequences for all 12 rice chromosomes were mapped on the molecular map with either or both of the probes, and flanking molecular markers on one or both sides were localized 0 to 8 cM away. The map locations of the centromere-associated markers corresponded very well with the positions of centromeric regions determined previously using trisomic analyses for 11 of the 12 chromosomes. The precise mapping of the centromeric regions using these probes makes the molecular map a more complete and informative tool for genomic studies, which will facilitate studies of the structure and function of the rice centromeres. The simplicity of this technique, together with the fact that these probes are also associated with the centromeric regions in other grass species, may provide a general approach to the mapping of centromeric regions in the genomes of other cereal crops.