Structural characterization of organic matter (OM) is crucial in assessing its role in soils and sediments. In this study, humic acids (HA) were sequentially extracted from a compost and characterized in terms of their molecular, chemical, and structural heterogeneity. HA was extracted in sequence a total of 28 times, using 0.1 mol L-1 Na4P2O7, and sequentially grouped into seven fractions. These fractions and the original compost were analyzed using UV-visible spectroscopy, C-13 nuclear magnetic resonance spectroscopy (NMR), diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), and an elemental analyzer. C-13-NMR and DRIFTS data indicated significant structural variations among the extracted HA fractions and the original compost. These data corresponded to a decrease in the atomic C/H ratio and to an increase in the atomic C/O ratio among these sequentially extracted HA fractions, which together represented an increase in aliphaticity in conjunction with a decrease in polarity and aromaticity. E-4/E-6 ratios indicated an increase in molecular size with increasing number of extractions. The structural variability of HA may significantly affect the sorption of organic pollutants and the reduction and complexation of metals by organic matter.