Source, transformation, and preservation mechanisms of dissolved organic matter (DOM) remain elemental questions in contemporary marine and aquatic sciences and represent a missing link in models of global elemental cycles. Although the chemical character of DOM is central to its fate in the global carbon cycle, DOM characterizations in long-term ecological research programs are rarely performed. We analyzed the variability in the quality of 134 DOM samples collected from 12 Long Term Ecological Research stations by quantification of organic carbon and nitrogen concentration in addition to analysis of UV-visible absorbance and fluorescence spectra. The fluorescence spectra were further characterized by parallel factor analysis. There was a large range in both concentration and quality of the DOM, with the dissolved organic carbon (DOC) concentration ranging from less than 1 mgC/L to over 30 mgC/L. The ranges of specific UV absorbance and fluorescence parameters suggested significant variations in DOM composition within a specific study area, on both spatial and temporal scales. There was no correlation between DOC concentration and any DOM quality parameter, illustrating that comparing across biomes, large variations in DOM quality are not necessarily associated with corresponding large ranges in DOC concentrations. The data presented here emphasize that optical properties of DOM can be highly variable and controlled by different physical (e.g., hydrology), chemical (e.g., photoreactivity/redox conditions), and biological (e.g., primary productivity) processes, and as such can have important ecological consequences. This study demonstrates that relatively simple DOM absorbance and/or fluorescence measurements can be incorporated into long-term ecological research and monitoring programs, resulting in advanced understanding of organic matter dynamics in aquatic ecosystems.