I-12CMS(2) and I-12CMS(3) are sugar beet lines with different sources of cytoplasmic male sterility (CMS) derived from wild beets in Turkey and Pakistan, respectively. We established that I-12CMS(2) has a genetically distinct cytoplasm, but its mitochondrial genome is very similar to I-12CMS(3). Male fertility was assessed in F-1 hybrids produced with a common pollen parent. Fertility in the F-1's carrying the I-12CMS(3) cytoplasm exceeded that of the F-1's with the I-12CMS(2) cytoplasm. Organization of the I-12CMS(2) and I-12CMS(3) mitochondrial genomes were compared based on their physical maps. Mitochondrial genomes of the two strains were largely collinear, except for a large deletion in the noncoding region of I-12CMS(2). Because a mitochondrial orf129 in the I-12CMS(3) cytoplasm is associated with a male sterility phenotype and preservation of orf129 was evident in I-12CMS(2), I-12CMS(2) orf129 was investigated in detail. I-12CMS(2) plants contained three to five times more ORF129 protein than did I-12CMS(3) plants. A single nucleotide substitution, present in the putative promoter region of orf129, appeared to be responsible for the differential accumulation of orf129 transcript. A long N-terminal extension of atp6 is a common feature of some beet CMSs and is found in I-12CMS(2), but the amino acid sequence is unique. I-12CMS(3) mitochondria, but not I-12CMS(2) mitochondria, were found to be heteroplasmic. This heteroplasmy is characterized by a substoichiometric DNA molecule(s) that has at least two I-12CMS(2)-type mitochondrial loci, suggesting the possibility that the I-12CMS(2) mitochondrial genome might have evolved from such a substoichiometric DNA molecule in I-12CMS(3) mitochondria.