Cucumber (Cucumis sativus) has one of the largest mitochondrial genomes known among all eukaryotes, due in part to the accumulation of short 20 to 60 bp repetitive DNA motifs. Recombination among these repetitive DNAs produces rearrangements affecting organization and expression of mitochondrial genes. To more efficiently identify rearrangements in the cucumber mitochondrial DNA, we built two nonoverlapping 800 and 220 kb BAC contigs and assigned major mitochondrial genes to these BACs. Polymorphism carried on the largest BAC contig was used to confirm paternal transmission. Mitochondrial genes were distributed across BACs and physically distant, although occasional clustering was observed. Introns in the nad1, nad4, and nad7 genes were larger than those reported in other plants, due in part to accumulation of short repetitive DNAs and indicating that increased intron sizes contributed to mitochondrial genome expansion in cucumber. Mitochondrial genes atp6 and atp9 are physically close to each other and cotranscribed. These physical contigs will be useful for eventual sequencing of the cucumber mitochondrial DNA, which can be exploited to more efficiently screen for unique rearrangements affecting mitochondrial gene expression.