The FTIR method was used to study the melting behavior of poly(ethylene 2,6-naphthalene-dicarboxylate) (PEN) samples of different thermal history of crystallization. With subtraction of the spectra of PEN samples of different crystallinities and amorphous PEN, the spectra of crystalline trans conformers, amorphous trans conformers, and amorphous gauche conformers were obtained. By following the changes in intensity of various gauche and crystalline bands during heating, the phenomenon of multiple melting endothermic peaks was shown to be due to the melting of imperfect crystalline conformation formed during crystallization. For two-step annealed samples, the crystalline conformational defects change into a more perfect one during annealing at higher temperatures.