The film forming behaviour and hydrophobicity of high amylose (HA) starch in the presence of three different natural waxes (beeswax, candelilla wax and carnauba wax) were studied in the presence and absence of Tween-80. The HA starch:glycerol (G) ratio was maintained at 80:20 (on dry solid basis) and the concentration of wax was varied from 5% and 10% (w/w). The melted wax samples were homogenized with HAG dispersion with or without Tween-80 and the films were prepared by solution casting. The hydrophobicity and water-barrier properties in these films were determined by using contact angle (CA), water vapour permeability (K
•Synchrotron-FTIR showed the distribution of wax and PEG-isocyanate across the film.•Wax and PEG-isocyanate distributions and film surface altered the film's hydrophobicity.•High contact angle occurred when PEG-isocyanate was concentrated in the middle of the film.•Hydrophobic films possessed hierarchical wax pinnacles and textured surface.•The wax pinnacles were 10μm in diameter and distributed very closely to each other. This study proposes a novel method for improving surface hydrophobicity of glycerol plasticized high amylose (HAG) films. We used polyethylene glycol isocyanate (PEG-iso) crosslinker to link HAG and three natural waxes (beeswax, candelilla wax and carnauba wax) to produce HAG+wax+PEG-iso films. The spatial distributions of wax and PEG-iso across the thickness of these films were determined using Synchrotron-based Fourier transform infrared spectroscopy. The hydrophobicity and surface morphology of the films were determined using contact angle (CA) and scanning electron microscopic measurements, respectively. The distribution patterns of wax and the PEG-iso across the thickness of the film, and the nature of crystalline patterns formed on the surface of these films were found to be the key factors affecting surface hydrophobicity. The highest hydrophobicity (CA >90°) was created when the PEG-iso was primarily distributed in the interior of the films and a hierarchical circular pinnacle structure of solidified wax was formed on the surface.