Rivers are a large source of dissolved organic matter (DOM) of terrestrial origin to the oceans and thus are important ecosystems that contribute to the global biogeochemical cycles. In these terrestrially influenced ecosystems, microbes depend on DOM and function as a metabolic force that decomposes and transforms DOM and are thus important contributors to the global carbon sequestration and export. Despite their importance, very little is known about the relationships between microbial metabolic capabilities and DOM composition found within and among river ecosystems. Here, we examined the microbial genetic diversity, metabolic potentials and gene expression in relation to the DOM composition in 5 major U.S. rivers over a seasonal cycle. We combined high-throughput metagenomics, metatranscriptomics and FT-ICR mass spectrometry of DOM to evaluate microbe-DOM interactions at the molecular level. Weighted co-occurrence networks revealed significant interactions between microbial gene expression and DOM chemistry, thus highlighting associations between key metabolic genes and biologically labile DOM molecules in river ecosystems. Overall, this study provides a highly resolved view of the potential for carbon mobilization by riverine microbes and highlights differing ecological roles in carbon turnover for the resident riverine microbial communities.