The mitochondrial–nuclear interaction is essential for cellular homeostasis and proper function. DNA methylation plays an important role in gene expression regulation, as well as in maintaining genomic stability. Isonuclear-alloplasmic and isoplasmic-allonuclear lines were used as materials in this study. The mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) from maize tassels were used for DNA methylation analysis with high-performance liquid chromatography. The results were as follows. (i) Different mtDNA methylation levels were detected within the isoplasmic-allonuclear lines and varied nDNA methylation levels were also detected among the isonuclear-alloplasmic lines. (ii) From the pollen mother cell stage to the binucleate stage, the change pattern of DNA methylation levels varied among cytoplasmic male sterility (CMS) lines CMS-C, CMS-T, and CMS-S (CMS Charrua, Texas, and USDA, respectively). The DNA methylation level peaked at the tetrad stage for the CMS-C line, at the mononuclear stage for the CMS-T line, and at the binucleate stage for the CMS-S line. Interestingly, the peaked periods for DNA methylation level just meet their critical abortive periods of CMS-C, CMS-T, and CMS-S lines. Finally, (iii) the change trends of methylation levels for both mtDNA and nDNA are highly consistent for every experimental material. Based on these results, we speculated that the epigenetic status affected by mitochondrial–nuclear interaction may be involved in the regulation of maize CMS.