Transmission electron microscopy (TEM) has been used to observe the lamellar melting and structural rearrangement processes on nanoscale for cold-crystallized isotactic polystyrene (iPS) spherulites. A series of morphological observations indicate that, not only the spherulites consist of two distinct crystal populations (i.e., the dominant and subsidiary lamellae) with different degrees of perfection as often suggested in the literature, but most interestingly, even along an individual lamella the perfection of the internal organization and consequently the thermal stability appear not uniform probably due to the uneven distribution of tacticity along polymer chains. These structural differences within spherulites and single lamellae are reflected by the lamellar fragmentation upon partially melting at different temperatures for the initially cold-crystallized iPS.