DNA methylation is one of the most important epigenetic modifications. It is vital to maintain the stability of the whole genome and regulate the expression of tissue-specific genes. Methylation level and distribution throughout genomic DNA are significantly correlated with gene expression rate. In order to investigate the changes of genomic DNA methylation level associated with heterosis of Epinephelus spp., we applied methylation-sensitive amplification polymorphism (MSAP) technology to detect the genomic DNA methylation levels of longtooth grouper (E. moara), giant grouper (E. lanceolatus), and their F1 hybrid [E. moara (♀) × E. lanceolatus (♂)], and we analyzed the difference of DNA methylation level between the F1 hybrid and parents. The results showed that overall genomic DNA methylation was higher in E. moara and E. lanceolatus. The total methylation rates of E. moara, E. lanceolatus, and the F1 hybrid were 60.62%, 59.38%, and 55.78%, respectively. The rates of fully methylated sites were 31.37%, 30.67%, and 29.27%, respectively, and the hemi-methylation rates were 29.25%, 28.71%, and 26.51%, respectively. The total methylation rate, the full methylation rate, and the hemi-methylation rate of DNA of the F1 hybrid were all significantly lower (P<0.01) than that of either parent. The full methylation rates were all higher than the hemi-methylation rates of E. moara, E. lanceolatus, and the F1 hybrid. The study also showed that the DNA methylation level of the F1 hybrid is negatively correlated with heterosis. Moreover, reduced DNA methylation level of the F1 hybrid may be one of the reasons for the heterosis phenomena such as rapid growth. In general, DNA methylation is negatively correlated with gene expression. The DNA methylation level of the hybrid was lower than either parent, allowing otherwise silenced genes to be activated, enhancing gene expression activity, and resulting in the hybrid progeny exhibiting heterosis. The relationship between methylation level of specific genes and heterosis, the genetic pattern of these DNA methylation sites, the effect on gene structure and function, and the regulatory mechanism require further study.