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Forage management and environmental conditions affect water soluble carbohydrate (WSC) storage, and, in turn, influence ruminant forage utilization in silvopastoral systems. The objective was to determine effects of four dependent variables: forage species [(non-native, C3 (orchardgrass (Dactylis glomerata L.)) and native C4 mix (8:1:1 big bluestem (Andropogon gerardii Vitman), little bluestem (Schizachyrium scoparium Michx. Nash) and indiangrass (Sorghastrum nutans L.))]; fertility (poultry litter and an unfertilized control); forage sampling date (mid-May, late-May, early-June, mid-June, and late-June); and hour of day (0800, 1100, 1400, and 1700 h) on WSC accumulation in a silvopasture. Concentrations of WSC (g kg DM−1) were greater (p ≤ 0.05) for C3 forages, with poultry litter not impacting WSC accumulation. Overall, WSC was greatest in mid-June, with the lowest WSC concentration observed at 0800 compared to 1100, 1400, and 1700 h (p ≤ 0.05). Therefore, harvesting forages later in the day resulted in greater WSC. A stepwise regression model indicated acid detergent fiber, ash, and forage P concentration were the best predictors (R2 = 0.85, p ≤ 0.05) of forage WSC. These results may be useful in future studies aimed at explaining diurnal cattle grazing preference and optimum forage harvest timing in silvopastoral systems.