© Crop Science Society of America. Cytoplasmic male sterility (CMS) has been exploited in the commercial pigeonpea [Cajanus cajan (L.) Millsp.] hybrid breeding system; however, the molecular mechanism behind this system is unknown. To understand the underlying molecular mechanism involved in A4 CMS system derived from C. cajanifolius (Haines) Maesen, 34 mitochondrial genes were analyzed for expression profiling and structural variation analysis between CMS line (ICRISAT Pigeonpea A line, ICPA 2039) and its cognate maintainer (ICPB 2039). Expression profiling of 34 mitochondrial genes revealed nine genes with significant fold differential gene expression at P £ 0.01, including one gene, nad4L, with 1366-fold higher expression in CMS line as compared with the maintainer. Structural variation analysis of these mitochondrial genes identified length variation between ICPA 2039 and ICPB 2039 for nad7a (subunit of nad7 gene). Sanger sequencing of nad4L and nad7a genes in the CMS and the maintainer lines identified two single nucleotide polymorphisms (SNPs) in upstream region of nad4L and a deletion of 10 bp in nad7a in the CMS line. Protein structure evaluation showed conformational changes in predicted protein structures for nad7a between ICPA 2039 and ICPB 2039 lines. All above analyses indicate association of nad7a gene with the CMS for A4 cytoplasm in pigeonpea. Additionally, one polymerase chain reaction (PCR) based Indel marker (nad7a_del) has been developed and validated for testing genetic purity of A4 derived CMS lines to strengthen the commercial hybrid breeding program in pigeonpea.