Poly [(R)-3-hydroxybutyrate] (P(3HB)) single crystals were grown isothermally at different crystallization temperatures (T(c)s) in a dilute solution. Structural changes of the sedimented single-crystal mats on heating were followed by real-time synchrotron small-angle X-ray scattering (SAXS) and wide-angle X-ray diffraction (WAXD) techniques, and the dependence of the annealing behavior on T-c was examined. The two-dimensional (2D) SAXS pattern clearly demonstrated that the P(3HB) single crystal exhibits a discontinuous increase in the lamellar thickness at an annealing temperature. The onset of the discontinuous thickening depended on T-c or the initial lamellar thickness. The discontinuous thickening occurred at higher temperature as the initial lamellar thickness increased. In the transition region where the discontinuous thickening progressed, the new scattering peak intensity increased while the original one decreased. On the other hand, the crystallinity, estimated from the 2D WAXD patterns, first decreased and then recovered. The lamellar thickening was accompanied by endo- and exothermic signals in differential scanning calorimetry. Accordingly, the discontinuous lamellar thickening is caused by partial melting and recrystallization. The degree of recovery in crystallinity fell with increasing initial lamellar thickness. This is most probably because the crystallization rate becomes slower with increasing temperature in the transition region. After the transition, the long period between the reorganized lamellae increased, while the crystallinity decreased and, finally, diminished due to complete melting.