Human longevity is always a biological hotspot and so much effort has been devoted to identifying genes and genetic variations associated with longer lives. Most of the demographic studies have highlighted that females have a longer life span than males. The reasons for this are not entirely clear. In this study, we carried out a pool-based, epigenome-wide investigation of DNA methylation profiles in male and female nonagenarians/centenarians using the Illumina 450 K Methylation Beadchip assays. Although no significant difference was detected for the average methylation levels of examined CpGs (or probes) between male and female samples, a significant number of differentially methylated probes (DMPs) were identified, which appeared to be enriched in certain chromosome regions and certain parts of genes. Further analysis of DMP-containing genes (named DMGs) revealed that almost all of them are solely hypermethylated or hypomethylated. Functional enrichment analysis of these DMGs indicated that DNA hypermethylation and hypomethylation may regulate genes involved in different biological processes, such as hormone regulation, neuron projection, and disease-related pathways. This is the first effort to explore the gender-based methylome difference in nonagenarians/centenarians, which may provide new insights into the complex mechanism of longevity gender gap of human beings.