Soybean is an important oilseed crop worldwide; however, it has a high sensitivity to temperature variation, particularly at the vegetative stage to the pod-filling stage. Temperature change affects physiochemical and genetic traits regulating the soybean agronomic yield. In this regard, the current study aimed to comparatively evaluate the effects of varying regimes of day and night temperatures (T1 = 20°C/12°C, T2 = 25°C/17°C, T3 = 30°C/22°C, T4 = 35°C/27°C, and T5 = 40°C/32°C) on physiological (chlorophyll, photosynthesis, stomatal conductance, transpiration, and membrane damage) biochemical (proline and antioxidant enzymes), genetic (GmDNJ1, GmDREB1G;1, GmHSF-34, GmPYL21, GmPIF4b, GmPIP1;6, GmGBP1, GmHsp90A2, GmTIP2;6, and GmEF8), and agronomic traits (pods per plant, seeds per plant, pod weight per plant, and seed yield per plant) of soybean cultivars (Swat-84 and NARC-1). The experiment was performed in soil plant atmosphere research (SPAR) units using two factorial arrangements with cultivars as one factor and temperature treatments as another factor. A significant increase in physiological, biochemical, and agronomic traits with increased gene expression was observed in both soybean cultivars at T4 (35°C/27°C) as compared to below and above regimes of temperatures. Additionally, it was established by correlation, principal component analysis (PCA), and heatmap analysis that the nature of soybean cultivars and the type of temperature treatments have a significant impact on the paired association of agronomic and biochemical traits, which in turn affects agronomic productivity. Furthermore, at corresponding temperature regimes, the expression of the genes matched the expression of physiochemical traits. The current study has demonstrated through extensive physiochemical, genetic, and biochemical analyses that the ideal day and night temperature for soybeans is T4 (35°C/27°C), with a small variation having a significant impact on productivity from the vegetative stage to the grain-filling stage.

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