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An excess of water-soluble carbohydrates (WSCs) can present a risk for horses prone to pasture-associated laminitis or some other metabolic conditions. Determining WSC concentrations in commonly grazed cool-season grasses, at different times of day and under different fertilization treatments, can help optimize grazing times and management strategies. The goals of this study were to develop a near-infrared reflectance spectroscopy (NIRS) calibration curve for WSC and to apply it to four cool-season grass species. Tall fescue, Kentucky bluegrass, orchardgrass, and perennial ryegrass (10 cultivars) were sampled every two to four weeks from plots with or without added nitrogen, in the morning and afternoon. WSCs were quantified colorimetrically for a sample subset, and these values were used to develop an NIRS calibration predicting WSC concentrations with 90% accuracy. An interaction of species, nitrogen treatment, time of day, and harvest date influenced WSC concentrations in 10 harvests (P =.040). A modest positive relationship was observed between photosynthetically active radiation and WSC concentration when morning and afternoon samples were included (r = 0.503; P =.024). On nine harvest dates, perennial ryegrass or tall fescue were highest in WSC. High-WSC cultivars included "Aberzest" and "Calibra" perennial ryegrass, "Ginger" Kentucky bluegrass, and "Bronson" and "Cajun II" tall fescue. Water-soluble carbohydrates did not exceed 150 g/kg freeze-dried weight, possibly due to assay method, sampling times, or defoliation. The results suggest that minimizing WSC intake for horses may be possible by cultivar choice, grazing time, or mowing frequency. • Water-soluble carbohydrates (WSCs) of 4 cool-season grasses were assayed. • Data were used for near-infrared reflectance spectroscopic (NIRS) calibration. • The NIRS equation was used to predict WSCs of 10 cool-season grass cultivars. • Water-soluble carbohydrates were lower in the morning than in the afternoon. • Cultivar choice and management may minimize equine WSC intake on pastures. [ABSTRACT FROM AUTHOR]