The hydrophilic or hydrophobic characteristics of dissolved organic matter (DOM) from different origins in lake and river waters were investigated using spectrometric and chromatographic analyses of water samples. DOM in a deep, mesotrophic lake (Lake Unmun) was fractionated using three types of ion exchange resins and classified into aquatic humic substances (AHS), hydrophobic neutrals (HoN), hydrophilic acids (HiA), hydrophilic neutrals (HiN), and bases (BaS). The DOM fractionation provided insight into the understanding of the nature of heterogeneous DOM molecules present in different water sources. The UV/DOC ratios were determined for samples from the influent river and lake waters during DOM fractionation and incubation. AHS prevailed over DOM in the lake and river waters. After biodegradation, the relative contribution of AHS in the total DOM became more significant. It indicates that the AHS fraction would increase while water stay long time in the lake.