The microstructure and thermal and mechanical properties of poly(lactic acid) (PLA) fibers after thermal treatment under both taut and free conditions at different temperatures were studied by differential scanning calorimetry, wide-angle X-ray diffraction, microscopy with polarized light, an acoustic method and tensile testing. In particular, the effects of thermal treatment conditions on crystallinity, molecular orientation, and mechanical properties, as well as their interrelationships, were investigated. Both the crystal orientation and crystallinity of the PLA fibers were higher after thermal treatment under taut conditions, even though the molecular orientation was not higher. Thermal treatment relaxed orientated segments, which resulted in increasing crystallinity; however, it did not indicate that the overall molecular alignment of the chains (molecular orientation) would necessarily be high. Tensile strength and elongation were increased with increasing treatment temperature up to 150°C, which corresponded to crystallinity and chain relaxation. The crystallinity detected by XRD, enthalpy measured by DSC, birefringence, and molecular orientation were compared to distinguish the effects of crystallinity, orientation of polymer chains, and crystals. POLYM. ENG. SCI., 2013. © 2012 Society of Plastics Engineers