Polydiacetylenes (PDAs), a family of highly p-conjugated polymers, have unique characteristics associated with their ability to self-assembly. Disruption of the extensively delocalized enyne backbones of molecularly ordered PDA side chains induces a blue-to-red color change, which has been elegantly applied in the design of chemosensors. Polydiacetylene (PDA) shows the blue PDAs with 640 nm maximum absorption wavelength, which can display a unique color change to a red with 550 nm maximum absorption wavelength upon environmental stimulations such as temperature, pH, metal ions, anions, surfactants, other biologically important molecules, etc. In addition, red-phase PDAs are fluorescent even though blue-phase PDAs are nonfluorescent, which enables PDAs to be not only colorimetric sensors but also fluorescent sensors. In fact, observations in colorimetric transitions have been suggested that the release of side chain strain taking place upon stimulation causes rotation about the C–C bonds in PDA backbone and then this conformational change perturbs the conjugated p-orbital array leading to a change in the chromophore responsible for the electronic transition. However, unfortunately, there is no experimental direct evidence regarding these long standing issues so that the exact mechanism for this process is not yet fully understood so far. Here we report real time snap shots dynamics of a new bisPDA with a well-packed naphthalene structure proved by in-situ glazing incidence Xray diffraction (GIXD) experiments under medium vacuum, displaying exceptional thermochromic reversibility with a clear blue to red colorimetric transition at elevated temperatures owing to the presence of unique hydrophobic interactions between alkyl chains as well as aryl moieties. Real-time observations of diffraction peaks of bis-PDA thin film as a function of temperature clearly reveal that molecular motions of PDA’s alkyl chains and naphthalene moieties play a crucial role in the colorimetric transition associated with the changes of the layer spacing, its coherence length, inter-chain distance of alkyl backbone, molecular tilt angle, and molecular packing of naphthalene moieties.