The release of dissolved organic matter (DOM) from agricultural land can have a large impact on the transport of N and phosphorus (P) to surface waters leading to water quality impairment. The speciation of DOM in agricultural grassland soils has received little attention thus far. Quantification of DOM speciation can improve our knowledge of its fate in these soils. Furthermore, the influence of temperature on DOM concentration and composition is still ambiguous. In this study, we determined the concentration and composition of water-extractable organic carbon (EOC), water-extractable organic N (EON), and water-extractable organic P (EOP) before and after incubation of sand, peat, and clay grassland soils at different temperatures (1.5 °C, 10 °C, and 20 °C) for 35 days. Extracted organic compounds were fractioned in three operationally defined fractions: humic acids (HA), fulvic acids (FA), and hydrophilic (Hy) compounds using a recently developed batch fractionation method. Both EON and EOP formed a major fraction of total N and P. Concentrations of EOC, EON, and EOP were different among the sand, peat, and clay soils, but their speciation was remarkably similar. The EOC and EON were mainly present in the hydrophobic form (HA and FA), whereas EOP was mainly present in the Hy fraction. An increase in temperature generally resulted in a decrease of the total EOC, EON, and EOP concentrations, whereas the speciation remained constant. The effect of temperature on the dynamics of DOM is not necessarily related to net changes in pool size of the HA, FA, and Hy fractions. Insight into the influence of incubation temperature on the dynamics of EOC, EON, and EOP can only be achieved when the processes responsible for the consumption and the production of dissolved organic nutrients are quantified.