In order to well understand the molecular basis of heterosis in soybean, the methylation-sensitive amplification polymorphism (MSAP) method based on capillary electrophoresis was used to estimate levels and patterns of cytosine methylation in 15-day post-emergence leaves of four parental lines [Jilin 47 (no. 19), EXP (no. 12), Jilin 38 (no. 3) and Yi 3 (no. 6)] and 12 hybrids [Jilin 38 x Yi 3(3 x 6), Jilin 38 x EXP(3 x 12), Jilin 38 x Jilin 47(3 x 19), Yi 3 x Jilin 38(6 x 3), Yi 3 x EXP(6 x 12), Yi 3 x Jilin 47(6 x 19), EXP x Jilin 38(12 x 3), EXP x Yi 3(12 x 6), EXP x Jilin 47(12 x 19), Jilin 47 x Jilin 38(19 x 3), Jilin 47 x Yi 3(19 x 6), Jilin 47 x EXP(19 x 12)]. In addition, 12 traits of the hybrids and their parents were also analyzed to understand the relationship between DNA methylation variation and heterosis. MSAP results showed that the total relative methylation level of all hybrids was lower than the corresponding middle parent value, indicating that the methylation degree was decreasing. And may express a variety of genes related to the phenotypic variation of hybridization. Moreover, the hemi-methylation levels of Jilin 38 x Jilin 47 and Yi 3 x Jilin 47 hybrids and full-methylation levels of EXP x Yi 3 and EXP x Jilin 47 hybrids was significant higher than the corresponding mid-parent values. In addition, the heredity of methylation from parents in hybrids is more than the variations, in which there were four types appeared great higher: A1, B4, B8, and D2. Furthermore, the results of relationship between genetic variation in DNA methylation and heterosis showed that the hypo-methylation had a promoting effect to increase node number, and the hype-methylation of hybrids was helpful to add to stem thick. Our results may provide new insights into well understanding the molecular mechanisms of heterosis at the epigenetic level in soybean.