Aggregate formation in conjugated polymer films is one of the most important phenomena thought to influence the photophysical properties of optical devices based on these materials. In the current work, we report the results of a detailed investigation on the morphology and chain aggregation dependence of optical gain in spin-coated thin films of the conjugated polymer poly[2-methoxy-5-(2′-ethylhexyloxy)-p-phenylene vinylene] (MEH-PPV). Extensive gain measurements are performed using the variable stripe length technique with picosecond pulse excitation. The polymer morphology and extent of aggregate formation in the films are controlled by thermal annealing, which is relevant to the fabrication and optimization of conjugated polymer-based optical devices. The aggregation state of the polymer chains increases with the annealing temperature, which results in a decrease in luminescence efficiency at low excitation density (≤1018 cm−3). However, the increase in aggregate formation with increasing annealing temperature does not significantly alter the optical gain; very large gain coefficients are still achieved in films containing a relatively large fraction of aggregates. Although the largest gain coefficients, 450 cm−1, are observed for as-cast (non-annealed) MEH-PPV films, very large gain coefficients of 315 and 365 cm−1 are also demonstrated for MEH-PPV films annealed at 60 and 80 °C, respectively, in spite of the enhanced packing morphology and conformational order of the polymer chains. These results are contrary to the commonly held view that aggregate formation has a detrimental effect on the amplified spontaneous emission behavior of polymer-based devices operating in the stimulated emission regime, as would be characteristic of lasers and optical amplifiers. Moreover, because aggregates promote favorable charge transport properties, our data have important implications for future development of electrically driven polymer lasers; improving carrier mobility through controlled increases in chain aggregation should provide a viable path for enhancing injection efficiency without significantly degrading optical gain.