The multiple melting behavior and morphologies of isotactic polystyrene (iPS) isothermally crystallized from the glassy state have been investigated by differential scanning calorimetry (DSC) and transmission electron microscopy (TEM). The combination of thermal analysis and morphological results indicates that two lamellar populations are responsible for the so-called double melting behavior in iPS. The low-temperature melting peak is attributed to the melting of less perfect (thinner or defect containing) subsidiary lamellae formed in the framework of the dominant (thicker or more perfect crystalline) lamellae upon isothermal crystallization. The high-temperature one is mainly due to the melting of the dominant thicker lamellae, and to some less extent, the melting of a recrystallized population coming from the melted defect lamellae during the heating process in DSC.