Horses spend a significant proportion of time grazing to meet their nutritional requirements. However, many domesticated horses now graze on energy-dense pastures designed to support production animals. Excessive consumption of these pastures that often contain significant quantities of water-soluble carbohydrates (WSC) has been linked to the development of pasture-associated laminitis. Although seasonal changes in pasture WSC content are well documented, the impact of fertilization or grass species composition are less defined. To evaluate the effect of season and fertilization on WSC concentrations, grassland plots were established at SRUC Aberdeen, UK. Six distinct cool-season seed mixtures (Table 1) were sown in triplicate plots (3m x 4m) and split in half to evaluate the effect of fertilizer treatment (285kg/ha of 21–8-11, annual application in March). Samples were collected on ice at set 6-to-8-week intervals (May–October) across 2 consecutive years. The Anthrone method was employed to estimate WSC content of lyophilised and milled samples, expressed as glucose and fructose equivalents [g/kg DM]. Independent t-test analysis (significance set at P < 0.05) revealed that fertilization had no overall effect on the WSC fraction for glucose (t(94) = 1.02, P = 0.99, 95% CI [-7.57, 23.51]) or fructose (t(94) = 1.28, P = 0.78, 95% CI [-4.69, 21.85]). Analysis of variance analysis demonstrated a significant effect of seed mixture (F = 2.56, P = 0.033 for glucose, F = 2.39, P = 0.044 for fructose) and season (glucose: F = 21.88, P = < 0.001, fructose:F = 21.09, P = < 0.001). WSC concentration was highest in samples collected in Spring, while the perennial rye grass mix (5) and a diverse herb mix (4) had the highest and lowest WSC contents across all samples, respectively. These preliminary results suggest that season and grass species composition have a greater effect on WSC concentrations in pasture than fertilizer treatment. Further studies are warranted to estimate the impact of fertilizer on other aspects of grass chemical composition that may have implications for managing pasture for grazing animals.