Atomic force microscopy coupled with infrared spectroscopy (AFM-IR) was combined with scanning electron microscopy (SEM) to investigate the tridimensional lamella assembly and polymer segregation mechanism at the nanoscale of equimolar crystalline/crystalline poly(3-hydroxybutyrate) (PHB) and poly(ethylene glycol) (PEG) blends, isothermally crystallized at different temperatures. The combination of solvent etching and thermal quenching-induced fractures of spherulites enabled the direct visualization of the lamella assembly and interior 3D spherulitic structure by SEM. Two different spherulitic patterns (banded and cone-like) were investigated after removing the PEG by ethanol etching. The banded spherulites are composed of perpendicularly oriented lamellae while flat-on lamellae are observed in the valleys. The transition ridge valley region contains right-handed, helically twisted lamellae. Cone-like spherulites are composed of highly ordered lamellae about 500 nm deep from the sample-air interface, and disordered structures are found deeper. The AFM-IR images and nanoscale infrared spectra reveal that the PEG components are mainly trapped into various interlamellae regions of PHB depending on the structure of the PHB crystal template. We report herein, for the first time, several tridimensional chemical images with full information on the diffusion of PEG in the PHB structure at the nanoscale.