The effect of the continuous application of an organic amendment (OA) on the accumulation and composition of humic acids (HAs) in various upland field soils was analyzed using a fractional precipitation technique. Fractionation patterns of HAs from plots treated with farmyard manure (FYM) or composted rice straw plus chemical fertilizer (CF) and from plots treated with CF alone were obtained by the step-wise addition of ethanol, 200–800 mL L−1, to HAs dissolved in 0.01 M sodium hydroxide (NaOH), and their degrees of humification were compared. HAs with a higher degree of humification precipitated in more dilute ethanol concentrations. The largest distribution shifted to a fraction that precipitated at a lower ethanol concentration with increasing degree of humification of bulk HAs, without an accompanying decrease in the amount of fractions with lower degrees of humification. A continuous OA application resulted in larger HA fractions with lower degrees of humification in most of the fields. The progression of humification in OA-derived HAs and interactions with indigenous soil HAs were also suggested from the fractionation patterns. The structural properties of the sub-fractions were evaluated using Miyagi soil (Cambisol) HAs. High performance size exclusion chromatography indicated that the larger molecular size molecules tended to precipitate at lower ethanol concentrations. The contribution of FYM-derived materials to the FYM+CF plot HA sub-fractions was suggested by a larger distribution of larger molecular size molecules compared to the corresponding HA sub-fractions from the CF plot soil. 13C cross polarization/magic angle spinning nuclear magnetic resonance analysis indicated that the fractions that precipitated at lower ethanol concentrations had more aromatic characteristics than the fractions that precipitated at higher ethanol concentrations and were soluble in 800 mL L−1 ethanol. These samples were characterized by a high alkyl carbon (C) content and intense signals corresponding to methoxyl and phenolic C. A correlation analysis for the HA sub-fractions both from the FYM+CF and CF plots indicated that their degree of humification corresponds to the composition of C functional groups.