A high-density genetic map was constructed with a RILs population (NJRIKY) from a cross of two cultivated soybean Kefeng 1 and Nannong 1138-2, using RFLP, SSR, RAPD and AFLP markers. The map consists of 24 linkage groups, which include 792 genetic markers, and covers 2,320.7 cM with an average genetic distance of 2.9 cM. The higher polymorphsims between two parents were identified with SSR markers. Due to its simplicity and stability, they could be used as anchored marker to integrate different maps constructed with different genetic markers. AFLP marker system is efficient for map construction while it easily form clusters to cause big gaps in the map. A total of 21.7% of markers was found with distorted segregation in the RILs population. The high-density molecular linkage map is fundamental for gene localization, comparative genomics, and QTL mapping of important agronomic traits.