We investigated the molecular orientation, crystallization mechanism, phase structure and transitions of aligned electrospun fibers, including the constrained amorphous phase and its relationship to the mesophase. Aligned poly(lactic acid) (PLA) fibers were successfully electrospun (ES) by adopting a highspeed rotating wheel as the counter electrode. Using thermal analysis and wide angle X-ray scattering (WAXS), we evaluated the confinement that exists in as-spun amorphous, and heat-treated semi-crystalline, fibers. Differential scanning calorimetry confirmed the existence of a constrained amorphous phase in as-spun aligned fibers, without the presence of crystals or fillers to serve as fixed physical constraints. Then, using WAXS, for the first time the mesophase fraction, consisting of oriented non-crystalline PLA chains, was quantitatively characterized in PLA nanofibers. The formation of oriented crystals during subsequent heating, and the evolution of the phase fractions (crystal, mobile amorphous, and constrained amorphous) with temperature, were investigated as to their dependence upon the method of fixing the ends of the fibers. In free-end ES nanofibers, the mesophase does not directly transition into the crystal phase of higher packing order. Instead, at temperatures above T-g, the free-end fiber undergoes residual stress relaxation, accompanied by the devitrification of the mesophase from a confined solid state to,a mobile liquid state. The mesophase, which possesses some degree of medium-range order, behaves very similarly to the rigid amorphous phase, and a correlation between the two is established in free-end fibers. On the other hand, for fixed-end as-spun fibers, the mesophase forms a more ordered structure when heated. By fixing the fiber ends, relaxation of the mesophase above T-g is partially disabled. The more ordered mesophase that survives above T-g undergoes a mesophase-to-crystal phase transition upon further heating, making an additional contribution to the heat of fusion. A new phase structure model is proposed to describe aligned electrospun PLA nanofibers. (C) 2013 Elsevier Ltd. All rights reserved.