Gametogenesis is associated with DNA methylation and involves complicated and delicate gene regulation network in which stem cell marker genes exert their functions. Therefore, it is necessary to investigate DNA methylation profiles of those genes in mature gametes that have an effect on embryo development. However, to date, there are limited data available on these genes in mature gametes of bovine. Here we show methylation profiles in 5' terminal regions of five pluripotency-related genes (Oct4, Sox2, Nanog, Rex1 and Fgf4) in bovine mature gametes, based on the reasoning that the five genes harbour CpG islands in their own 5' terminal regions, which are frequently the targets of DNA methylation. The results showed that Oct4 and Fgf4 exhibited significant hypermethylation in sperm compared with that in oocytes (p < 0.01), while Sox2 and Nanog displayed relatively the same methylation levels between sperm and oocytes (p > 0.05). Additionally, Rex1 showed a relatively high methylation level in sperm than in oocytes, although no significant differences were found (p > 0.05). In conclusion, bovine mature gametes exhibited two methylation profiles in terms of the five genes, one being non-sex-specific and the other being sex-specific.