Root traits (RTs) of soybean (Glycine max (L.) Merr.) that can be improved through long-term genetic breeding have been identified. However, whether resistance to environmental stresses can be enhanced and more detailed information on the relationships between RTs and seed yield remain unclear. Here, we used a pot-culture experiment with 13 varieties released in different years to investigate the changes in some RTs resulting from genetic breeding-based improvements. We determined whether RTs in different varieties respond to increasing fertilization rates (FRs) differently and quantified the contributions of RTs to seed yield variation among varieties. Decades of genetic selection have resulted in significant desired changes in RTs as well as the seed yield (per plant) under different FR conditions. The RT values of soybean receiving the 1.1 g pot−1 FR treatment increased significantly by 8.20%, 8.75% and 8.68%, whereas those receiving the 2.2 g pot−1 FR treatment decreased by 14.31%, 13.28% and 5.52%, for old, middle and new variety groups, respectively, compared with the no fertilizer treatment, indicating that the tolerance of root to fertilizer stress was enhanced. The results of artificial interference analysis showed that root length at the full bloom stage, root-to-shoot ratio at the full seed stage and root activity at the beginning maturity stage were the most important factors affecting seed yield, contributing approximately 54%, 58% and 59%, respectively, to seed yield variation. Overall, our work provides a theoretical basis for future breeding, suggesting a direct selection of soybean RTs to improve soybean yield.