Background: Cytoplasmic male sterility (CMS) is a trait of economic importance in the production of hybrid seeds. In CMS-S maize, spontaneous revertants appear frequently in field-grown female populations where only male-sterile plants were expected. Loss of sterility conferring regions or other rearrangements in the mitochondrial genome were reported to associate with these reversions. However, the relationship about mitochondrial function and sterility stability is largely unknown. Results: Here we reported different spontaneous reversion rates between two CMS-S subtypes, one with high rate of reversion to fertility (CMS-Sa) and another with more stable sterility (CMS-Sb). Through next-generation sequencing, we assembled and compared mitochondrial genomes of two CMS-S subtypes. Phylogenetic analysis revealed strong similarity between two mitochondrial genomes. The sterility associated regions, S plasmids, and terminal inverted repeats (TIRs) were intact in both. Two subtypes maintained high level of sterility gene orf355 transcription in anther tissue. Majority of the functional genes are identical in both amino acid and nucleotide sequence in both subtypes, except for that of NADH dehydrogenase subunit 1 (nad1). In the mitochondrial genome of CMS-Sb, 3.3-kilobase sequence was lost in the 17-kb repeat region, leading to the elimination of one copy of nad1 exon1. Consequently, there are two copies of nad1 exon1 in CMS-Sa, but only one copy in CMS-Sb. During pollen development, obvious induction of nad1 transcription and mitochondrial biogenesis were observed in anther of CMS-Sa, but not in anther of CMS-Sb. We suggested that the impaired mitochondrial function in anther of CMS-Sb is associated with the enhanced sterility stability.Conclusions: Comprehensive analysis revealed the sequence diversity in copy number of mitochondrial encoding gene nad1 in two subtypes of CMS-S maize. This copy number shift affects the gene transcription and organelle biogenesis of anther tissue, and is associated with different spontaneous sterility reversion. Our study suggests the mechanism that the robustness of mitochondrial function underlying the sterility stability of CMS-S maize.