In recent years, breeding for hybrid cultivars of soybean [Glycine max (L.) Merr.] for utiliazation of heterosis has been paid great attention, but there are few reports on the fundamental aspects regarding the heterosis in soybeans.. In fact, for a real utilization of hybrid soybean, the important prerequisite is high heterosis. Therefore, a fundamental effort in hybrid breeding is the choice of parents and identification of superior hybrid combinations. In this paper, heterosis and combining ability were determined using eight key parental materials, including seven from Huang-Huai region and one from US as well as their 28 F1 diallel crosses in Huai’an, Jiangsu, China from 2003 to 2005, and relationships between F1 performance and its pedigree-based and SSR-based genetic distances were investigated. The results showed that there were heterobeltiosis in yield among parents in Huang-Huai region with the average heterobeltiosis of 20.39%, and a large difference among hybridized combinations with a range from ?5.34% to 76.88%. We screened combinations, among them, Yudou 22 × Jindou 27, Huaidou 4 × Jindou 27, and Youbian 30 × Meng 9024 had the heterobeltiosis in yield of 76.88%, 29.90%, and 34.42%, respectively. Among these parents used above, Youbian 30 and Jindou 27 were the elite. Heterosis of pods per plant and seeds per plant were relatively in accord with yield heterosis. Yield heterosis in parents was related to general combining ability (GCA) and specific combining ability(SCA). One of parents has high GCA or both have high GCA and high SCA in high-yield combinations. Parents-based cluster and SSR-based cluster analysis revealed that genetic relationships for eight parents were basically consistent, and eight parents were classified into two groups, one including six varieties from middle and south of Huang-Huai region, the other consisting of one from Shanxi and one from America. Therefore, certain genetic distance is require for a cross with high heterosis and high yield, but genetic distance is not an only determinant factor for high-yield heterosis.