The Columbia River is the largest North American river which discharges into the Pacific Ocean and by that exports large quantities of dissolved organic carbon (DOC). In such riverine environments, terrestrially derived organic matter is a major DOC constituent and also a part of colored dissolved organic matter (CDOM). The optical properties of CDOM have previously been utilized to quantify the total dissolved organic matter (DOM) dynamics with in situ sensors. However, the validity of CDOM as quantitative proxy depends on the quality of correlation between DOC and CDOM, which has been disputed for the Columbia River. Therefore, the aim of this study was to examine the optical properties of CDOM, to investigate the seasonal variability of DOM, and to characterize its molecular composition. Furthermore, molecular imprints of primary production were investigated. From March to August 2013, 78 samples were collected at two stations in the lower Columbia River (near Portland, OR). Both stations were equipped with in situ sensors to record physical and biogeochemical data. The samples were characterized by fluorescence and absorbance analyses. On four occasions, DOM was solid-phase extracted to analyze its molecular composition using Fourier transform ion cyclotron resonance mass spectrometry. Based on the sensor data, a spring phytoplankton bloom and the spring freshet were identified. During July and August 2013, solar radiation depleted in situ CDOM fluorescence, but not absorbance. Contrary to previous studies, absorbance was identified as a valid proxy for DOC in the Columbia River (R2 =0.9). Using in situ sensor data, the estimation of DOC concentrations during the study period was achieved within a range of 33 μM. The phytoplankton bloom coincided with a shift of the fluorescence index (R2 =0.7), while changes in the freshness index correlated with higher DOC concentration during the spring freshet (R2 =0.8). The correlation between FT-ICR mass peaks and in situ parameters allowed an distinction of allochthonous and autochthonous molecules, thus elucidating the fluorescence observations on a molecular level. The results of this study are the basis for a more accurate in situ monitoring of DOM dynamics. Spectroscopic and spectrometric analyses illustrated the seasonal variability of DOM and exposed molecular imprints of primary production on the Columbia River DOM.