Polydiacetylenes (PDAs) possess dramatic chromatic transitions, which can be exploited to make stimuli-responsive sensors. Key to developing robust sensors is understanding the changes in molecular structure responsible for these chromatic transitions. We report systematic multiscale studies of different surfactant PDAs using grazing incidence X-ray diffraction (GIXD), X-ray reflectivity (XRR), and atomic force microscopy (AFM) to determine the structure of PDAs in different phases. Monomer films have significant multilayer registry, which is lost after polymerization to the blue phase. Still the monomer and the blue phase are structurally similar: the molecules are uniformly tilted with the diacetylene motif aligned to enable topochemical polymerization. The transition from the monomer to the blue to red phase PDA is not uniform, and at the molecular scale, all three phases may be present within a film. Measurements with Zn²⁺ in the subphase or boronic acid functionalized PDA precluded the formation of the red phase, enabling blue phase PDA to be characterized unambiguously. In the blue phase, the molecules are uniformly tilted without registry between multilayers. In comparison, red phase PDA adopts a nonplanar conformation with a decrease in molecular tilt. Red phase GIXD patterns can be fit with two potential conformations, a kinked geometry or a twisted geometry. Finally, AFM and XRR measurements reveal that even relatively uniform appearing PDA films are heterogeneous, from no film (bare) regions to monolayer, bilayer, trilayer, and many multilayers at boundaries.