We have constructed a physical map of the mitochondrial DNA of a cytoplasmic male sterile (CMS) sugar beet line, TK81-MS, and compared it with that published for normal fertile sugar beet (cv. TK81-O) to clarify the differences between the CMS and normal mitochondrial genomes. The TK81-MS genome is present as a single circular molecule of 481.8 kb, or as two molecules of 184.9 and 296.9 kb. The CMS genome was found to be highly rearranged relative to the normal mitochondrial genome, with at least fifteen rearrangement and/or inversion events being required to align the two DNAs. Analysis of transcription patterns of known mitochondrial genes and rearranged regions revealed six genes, coxI, coxII, atpA, atp6, rps3, and orf324, whose expression is altered in the CMS line relative to the normal line. Of these six, only the coxI transcript pattern differs between male-sterile and fertility-restored genotypes, making it likely that the coxI locus is involved in mediating CMS in sugar beet.