The technique of scanning microbeam small-angle X-ray scattering (SAXS) has been applied to investigate the inhomogeneity in the nanostructure of a single bilateral wool fibre in relation to two types of cortices, the so-called orthocortex and paracortex, which are located at the outer side and the inner side of the curved fibre, respectively. On the basis of the equatorial scattering intensity profiles from both experiments and simulation, the inhomogeneity in the mean IF (intermediate filament)–IF distance, IF diameter and IF orientation, corresponding to the structural difference between the orthocortex and the paracortex in bilateral wool fibre, has been quantitatively obtained.