This study investigated the characteristics of natural organic matter (NOM) with tXAD resin and FT-IR in the Yeongsan river system of Gwangju region. NOM fractionation by XAD 8/4 resins was used to classify hydrophobic and hydrophilic substances. FTIR was applied to classify functional groups in the structure of NOM. In the XAD investigation, most of the four site-samples were mainly hydrophilic substances. In March, hydrophilic substances were dominant in the Gwangju 1 site (GJ-1), while hydrophilic substances were dominant for the other sites. In May, samples of all four sites were hydrophilic with a vigorous activity of microorganism due to increasing temperatures. The October results were very similar with those from March. In the FT-IR investigation, most of the broad and large peaks were assigned to the aliphatic group, particularly the OH group, C-H, C-H2, C-H3, and C-O alcohol group. All were related to hydrophilic substances. Other peaks showed the aromatic group, particularly the C=O (Ketone) Group. As a result, there is an identification of NOM in the Yeongsan river system composing mainly of hydrophilic substances and functional groups (OH, C-H etc.) of the aliphatic compound.