Soybean is vital for global food security, and understanding its response to environmental changes is crucial. We examined the interannual variation in seed traits of 14 short-season soybean cultivars from seven decades (1932-1992) in Eastern Canada, using field trials data from 1993 to 2016. Impacts of growing season weather variables—precipitation, mean maximum temperature (MTemp), and mean maximum vapor pressure deficit (MVPD), as well as historical atmospheric CO ₂—on seed yield, protein and oil percentages, carbon isotope discrimination (Δ ¹³C), nitrogen isotopic composition (δ ¹⁵N) were assessed. Seed yield and Δ ¹³C increased with precipitation but decreased with MTemp and MVPD. Seed carbon percentage and Δ ¹³C increased with atmospheric CO ₂, while seed protein and oil percentages, and δ ¹⁵N decreased. Hierarchical partitioning highlighted vulnerability of soybean yield during the early reproductive stages (R1-R3, July) as well as the protein yield during the pod-formation and seed-filling period (R4-R6, August). Historical cultivar selection favored seed and oil yields, but not protein yield, Δ ¹³C, and δ ¹⁵N. Correlations between Δ ¹³C, δ ¹⁵N, and seed yield suggest selecting for higher yield may indirectly reduce water-use efficiency (indicated by higher D ¹³C) and enhanced biological nitrogen fixation (reflected by lower d ¹⁵N).