The completed rice genome sequence will accelerate progress on the identification and functional classification of biologically important genes and serve as an invaluable resource for the comparative analysis of grass genomes. In this study, methods were developed for sequence-based alignment of sorghum and rice chromosomes and for refining the sorghum genetic/physical map based on the rice genome sequence. A framework of 135 BAC contigs spanning approximately 33 Mbp was anchored to sorghum chromosome 3. A limited number of sequences were collected from 118 of the BACs and subjected to BLASTX analysis to identify putative genes and BLASTN analysis to identify sequence matches to the rice genome. Extensive conservation of gene content and order between sorghum chromosome 3 and the homeologous rice chromosome 1 was observed. One large-scale rearrangement was detected involving the inversion of an approximately 59 cM block of the short arm of sorghum chromosome 3. Several small-scale changes in gene collinearity were detected, indicating that single genes and/or small clusters of genes have moved since the divergence of sorghum and rice. Additionally, the alignment of the sorghum physical map to the rice genome sequence allowed sequence-assisted assembly of an approximately 1.6 Mbp sorghum BAC contig. This streamlined approach to high-resolution genome alignment and map building will yield important information about the relationships between rice and sorghum genes and genomic segments and ultimately enhance our understanding of cereal genome structure and evolution.