Time-dependent infrared (IR) spectral variations during isothermal melt-crystallization process of poly(3-hydroxybutyrate) (PHB) have been analyzed for different wavenumber regions (C-H, C=O, C-O-C, and C-C stretching vibration regions) by difference spectra, second derivatives, and two-dimensional (2D) correlation analysis, and the bands characteristic of crystalline and amorphous parts are identified in each region. By the 2D correlation analysis, it has been found that the intensity changes in the 1731 cm(-1) band, which may be due to the intermediate state, and in the 1722 cm(-1) band due to the crystal packing occur out of phase with each other, whereas those in the two amorphous C=O stretching bands (1747 and 1739 cm(-1)) ascribed to the different conformations of the main chain are synchronous, which represents a cooperative conformational rearrangement for the C=O groups in the amorphous state during the crystallization process. Meanwhile, a distinct delay between the intensity change rates of bands at 1184 and 825 cm(-1) indicates that the adjustment of C-O-C backbone occurs faster than that of C-C backbone in PHB. This result has also been confirmed by investigating the 2D correlation spectra of PHB in various spectral regions. On the basis of these observations, a physical picture on the molecular evolution of PHB during the melt-crystallization process has been derived.