New information on the chemical composition of dissolved organic matter (DOM) in three lakes in Minnesota has been gained from spectral editing and two-dimensional nuclear magnetic resonance (NMR) methods, indicating the effects of lake hydrological settings on DOM composition. Williams Lake (WL), Shingobee Lake (SL), and Manganika Lake (ML) had different source inputs, and the lake water residence time (WRT) of WL was markedly longer than that of SL and ML. The hydrophobic organic acid (HPOA) and transphilic organic acid (TPIA) fractions combined comprised >50% of total DOM in these lakes, and contained carboxyl-rich alicyclic molecules (CRAM), aromatics, carbohydrates, and N-containing compounds. The previously understudied TPIA fractions contained fewer aromatics, more oxygen-rich CRAM, and more N-containing compounds compared to the corresponding HPOA. CRAM represented the predominant component in DOM from all lakes studied, and more so in WL than in SL and ML. Aromatics including lignin residues and phenols decreased in relative abundances from ML to SL and WL. Carbohydrates and N-containing compounds were minor components in both HPOA and TPIA and did not show large variations among the three lakes. The increased relative abundances of CRAM in DOM from ML, SL to WL suggested the selective preservation of CRAM with increased residence time.