The structural variance of polydiacetylene (PDA) at the nanoscale level, even under the same fabrication conditions, is one of the origins of its poor reproducibility in chemo/biosensing. In this work, we present a spatial map of such structural distributions within a single crystal by taking advantage of the recent development of hyperspectral microscopy at visible wavelengths. Hyperspectral microscopy provides the distribution of absorption spectra at the spatial resolution of standard optical microscopy. By tracking the blue-to-red transition via this technique, we found that heat or pH stimulation leaves a unique pattern in the transition pathways.