Flooding is a serious problem for soybean production in eastern and southern China. Development of cultivars with tolerance to waterlogging is one of the effective ways to cope with the stress problem. For which, the genetic knowledge of waterlogging tolerance is of essential importance. The present study was aimed at revealing the inheritance and mapping the QTLs for waterlogging tolerance of soybean at seedling stage. The materials used were 175 recombinant inbred lines (RILs) derived from the cross Su88-M21 (Tolerant) × Xinyixiaoheidou (Highly sensitive), designated as NJRISX. A pot experiment was held under 5–7 cm waterlogging stress for 20 days at V2 stage. Based on the correlation and principal component analysis of eight traits, we calculated the joint waterlogging tolerance index from the average of individual tolerance index of plant height increment, number of leaves at the end of waterlogging and plant height at maturity to evaluate the tolerances of the materials. There existed obvious transgressive segregation and significant differences among the RIL lines. The segregation analysis under major gene plus polygene mixed inheritance model showed that waterlogging tolerance of soybean was controlled by two linked major genes plus polygenes with major gene heritability of 62.83% and polygene heritability of 8.90%. By using composite interval mapping (CIM) and multiple interval mapping (MIM) of WinQTL Cartographer Version 2.5, two QTLs conferring waterlogging tolerance were mapped in the marker regions of Satt229–Satt527 and Satt527–satt286 on linkage groups L2, explaining 11.76% and 12.34% of the total phenotypic variation, respectively. Accordingly, the results from segregation analysis and QTL mapping are relatively consistent in NJRISX population.