The link between composition and reactivity of dissolved organic matter (DOM) is central to understanding the role aquatic systems play in the global carbon cycle; yet, unifying concepts driving molecular composition have yet to be established. We characterized 37 DOM isolates from diverse aquatic ecosystems, including their stable and radiocarbon isotopes (δ¹³C-dissolved organic carbon (DOC) and Δ¹⁴C-DOC), optical properties (absorbance and fluorescence), and molecular composition (ultrahigh resolution mass spectrometry). Isolates encompassed end-members of allochthonous and autochthonous DOM from sites across the United States, the Pacific Ocean, and Antarctic lakes. Modern Δ¹⁴C-DOC and optical properties reflecting increased aromaticity, such as carbon specific UV absorbance at 254 nm (SUVA₂₅₄), were directly related to polyphenolic and polycyclic aromatic compounds, whereas enriched δ¹³C-DOC and optical properties reflecting autochthonous end-members were positively correlated to more aliphatic compounds. Furthermore, the two sets of autochthonous end-members (Pacific Ocean and Antarctic lakes) exhibited distinct molecular composition due to differences in extent of degradation. Across all sites and end-members studied, we find a consistent shift in composition with aging, highlighting the persistence of certain biomolecules concurrent with degradation time.