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The characterization of dissolved organic matter (DOM) is an important parameter for the management of water resources. In this study, physical and chemical characteristics of DOM in Kushiro river, and its territory (Eastern Hokkaido, Japan) were analyzed using multiple analytical tools. Multiple samples were collected from diverse locations (Kushiro river, Kushiro wetland, compost fertilizer, forest and treated sewage) and compared them against their DOM characteristics. Among the samples, Kushiro wetland samples presented higher specific ultraviolet absorbance (SUVA) values demonstrating DOM derived from vascular plants which are rich in aromatic-components. The SUVA values of the two samples of the Kushiro river were relatively close to those of the wetland samples implying the fact that the river contains high amount of plant derived aromatic DOM compounds, originating from the wetland. The excitation–emission matrix (EEM) fluorescence results showed that generally all water samples have two peaks namely, A and C. These two peaks are related to humic-like substance typically derived from the breakdown of organic matters. The excitation–emission matrix (EEM) fluorescence results of the river samples were also similar to those of the wetland samples, characterized by no appearance of protein, amino acid, polysaccharide, and fluvic like matter, but more pronounced to the aromatic component rich humic acid. Principal component analysis revealed that approximately 88% of the variance in the DOM characteristics might be explained by the type of different sources. From the loadings on PC1 and PC2 in the scores plot, the properties of the DOM of the wetland appeared to be similar to those of the river DOM. Combined results exhibited that the quality of the Kushiro river water is fundamentally inherited from the Kushiro wetland, and the main structure of the river water is characterized by poly-aromatic humic like matter and higher molecular weight.