In this study, we prepared two types of drawn films of poly[(R)-3-hydroxybutyrate], a microbial polyester: those containing only α-crystals and those containing both α- and β-crystals. Real-time wide-angle X-ray diffraction (WAXD) and real-time small-angle X-ray scattering (SAXS) measurements were performed during heating using synchrotron X-rays, along with high-speed differential scanning calorimetry (DSC), to measure the melting temperature of the β-crystals, which were present in a small amount. The changes induced in the highly ordered structure of the films were also observed. Based on the results of the real-time WAXD measurements, the melting temperature of the β-crystals was found to vary from 90 °C to 130 °C. The results of high-speed DSC supported these results. The real-time SAXS measurements allowed the changes in the highly order structure of the films to be observed. A model to explain the changes in the highly order structure is constructed based on the obtained results.