Abstract High-pressure size exclusion chromatography (HPSEC) has proven to be an effective method for determining the molecular weights (MW) of humic substances (HS) from a variety of aquatic and terrestrial environments. HPSEC systems often use a variable wavelength UV-vis detector, which detects the analytes based upon their chromophoric composition. HS contain a range of moieties with chromophores having unique molar absorptivities (for a given wavelength), and the calculated MW may be dependent upon the wavelength chosen for the analysis. As a consequence, the choice of wavelength becomes an important parameter for the reliable determination of MW by HPSEC. The effect of UV-vis detector wavelength on the determination of the MW distribution of selected humic and fulvic acids by HPSEC is examined in this paper. For the HS examined, both the number ( M n) and weight average ( M w) MW increased with increasing wavelength. The relative increase in MW was most pronounced for Lake Fryxell fulvic acid, with a 63 and 21% increase in M n and M w, respectively, between 220 and 380 nm. The increases observed for Suwannee River humic and fulvic acids were less pronounced. M n was more sensitive to changes in detector wavelength than M w, and as a result the target HS appeared to be less polydisperse at higher wavelengths. Within the range of wavelengths commonly used for the determination of MW of HS by HPSEC (i.e., 220–280 nm), the magnitude of the increases in MW was not significant compared to variability in MW that results from changes to other operational parameters in HPSEC.