Purpose The fractionation of soil humic acids (HAs) according to their hydrophobicity is a common procedure in the study of this polydispersed complex natural mixture, so that reversed-phase high-performance liquid chromatography (RP-HPLC) is used resulting in humic components of differing hydrophobic/hydrophilic properties. However, a comparative study of the hydrophobicity of fractions isolated from different soil HAs have not been addressed so far. Materials and methods The RP-HPLC with online absorbance detection was used for analysis of International Humic Substances Society soil standard HAs, chernozem soil HAs, and their electrophoretic fractions A, B, and C + D, obtained by tandem size exclusion chromatography-polyacrylamide gel electrophoresis. The strong relationship between hydrophobicity, electrophoretic mobility (EM), molecular size (MS), specific absorbance at 280 nm and aromaticity of HAs fractions was found. Results and discussion Independently of soil HAs genesis fraction A with lowest EM and highest MS is essentially more hydrophobic (60-73 % of the fraction amount remained adsorbed on the RP column) than medium EM and MS fraction B (33-47 % of the fraction amount remained adsorbed on the RP column). The lowest hydrophobicity belongs to fraction C + D with highest EM and lowest MS. Conclusions The most hydrophilic aromatic fraction C + D seems to have been bound with other mostly aliphatic hydrophobic fractions A and B through non covalent (possibly hydrogen) bonds. These data could be relevant to better understanding the overall makeup of soil HAs and their structural organization.