Kenaf is a great source of bast fiber and possesses significantly industrial interests. Cytoplasmic male sterility (CMS) is the basis of heterosis utilization in kenaf. Chloroplast, an important organelle for photosynthesis, could be associated with CMS. To understand the phylogenetic position and molecular basis of kenaf CMS from the perspective of chloroplast, the chloroplast (cp) genomes of the CMS line P3A and its maintainer line P3B were characterized and their comparative analysis was also performed. In this study, the chloroplast genomes of P3B and P3A were sequenced with 163,597 bp and 163,360 bp in length, respectively. A total of 131 genes including 85 protein coding genes (PCGs), 38 transfer RNA (tRNA) genes, and 8 ribosome RNA (rRNA) genes were annotated in P3B, while 132 genes containing 83 PCGs, 41 tRNA genes, and 8 rRNA genes were found in P3A. The phylogenetic tree revealed that kenaf was closely related to Hibiscus syriacus and Abelmoschus esculentus. Further analysis of single nucleotide polymorphism (SNP) and insertion and deletion (InDel) showed that compared with P3B, a total of 22 SNPs and 53 InDels were detected in gene coding region, gene intron, and intergenic regions of P3A. Remarkably, a total of 9 SNPs including 6 synonymous SNPs and 3 nonsynonymous SNPs were found in psbK, atpA, rpoC2, atpB, rpl20, clpP, rpoA, and ycf1. The present study provided basic information for further study of kenaf CMS mechsnism.