Abnormal programmed cell death in the tapetum is induced by reactive oxygen species (ROS), which are the main factors leading to cytoplasmic male sterility (CMS). These abnormalities are caused by genetic interactions between nuclear and cytoplasmic genes. To explore the role of chloroplast genes in ROS metabolism, next-generation and single-molecule real-time sequencing of the chloroplast genome were performed in the cotton CMS line Jin A (Jin A-CMS). Our results showed that the chloroplast genome is 160,042 bp in length and consists of 131 genes, including 112 functional genes. An analysis of the functional annotation and sequence comparison with the Gossypium hirsutum chloroplast genome as a reference revealed that 29 genes in Jin A-CMS have single-nucleotide polymorphisms, including subunits of ATP synthase, NAD(P)H-quinone redox reductase, and photosystem complexes. Compared to the Jin B maintainer, the anthers of Jin A-CMS at the microspore abortion stage have significantly lower expression of atpB, atpE, and atpF. The relative expression of these genes is significantly higher in the three-line F1 hybrids compared to Jin A-CMS. The ROS levels in the leaves increased in response to the silencing of atpE and atpF in cotton plants. In summary, the results of our study show that the ATP synthase subunit genes atpE and atpF are closely linked with ROS metabolism. These results provide basic information for the functional analysis of ATP synthase in cotton.