This study compared the sequence variations and expressions of 12 chloroplastic and 8 mitochondrial genes in three pepper (Capsicum annuum L.) cytoplasmic male sterile (CMS) lines, their maintainers and two control cultivars. The results showed that the three CMS lines were highly similar in chloroplastic and mitochondrial fragment sequences, with average similarities of 96.81 and 98.73 %, respectively, and their chloroplastic trnH–psbA intergenic spacer, photosystem II 47 kDa protein (psbB) genes, mitochondrial apocytochrome b (cob) and RNAD fragments have 1, 9, 8 and 7 distinct sites from the maintainer lines, respectively, and could be used as informative sites to distinguish CMS lines from the maintainer lines. Meanwhile, the expressions of mitochondrial cob, RNAD and pvs in the reproductive organs (flowers) of CMS lines are different from those of the maintainer lines, but their expressions in the vegetative organs (roots and leaves) are similar. The results indicate that cytoplasmic DNA polymorphisms are rare in CMS lines, and mitochondrial cob, RNAD and pvs genes are closely related to pollen abortion.