DNA methylation is an epigenetic modification, which is important for gene expression regulation. Although genome-wide DNA methylation studies have been reported in several plant species, the difference in the methylation pattern between the cytoplasmic male sterile (CMS) line and its maintainer in soybean remains unclear. We compared genome-wide DNA methylation between the soybean CMS line NJCMS1A and its maintainer NJCMS1B using methylated DNA immunoprecipitation combined with high-throughput sequencing (MeDIP-seq) technology. The results showed that the methylation level was higher in transposable elements (TEs) than promoter and intron; however, the methylation levels varied among different types of TEs with the highest level for long terminal repeats (LTRs) and the lowest for transcription start sites (TSS) and transcription termination sites (TTS). We observed 178 differentially methylated genes (DMGs) between NJCMS1A and NJCMS1B, including 156 hypomethylated and 22 hyper-methylated genes in NJCMS1A compared to NJCMS1B. Gene Ontology (GO) analysis showed that 114 DMGs were annotated to one or more GO categories, among which four GO terms were significantly enriched. KEGG pathway analysis showed that 18 DMGs were significantly enriched in 10 metabolism pathways, including homologous recombination, purine metabolism, proteasome, non-homologous end-joining, and pyrimidine