Cotton (Gossypium hirsutum) is often planted under sub-optimal early season temperatures, yet it is unknown whether this increases susceptibility to acute high temperature exposure. To address this, cotton seedlings were grown under optimal (30/20 °C) and sub-optimal (20/15 °C) growth temperature regimes, and comprehensive fluorescence analysis was conducted for cotyledons from both temperature regimes in response to incubation temperatures ranging from 30 to 50 °C. Results indicated that low growth temperature did not alter reaction center density (RC/CS0), but increased antenna size (ratio of antenna chlorophyll to reaction center chlorophyll; ABS/RC) and decreased the amplitude of the I to P phase of the fluorescence transient [ΔVIP; associated with the photosystem (PS) I content]. Photosynthetic performance indices (PItotal and PIABS), quantum yield of energy trapping (φPo), the contribution of the thylakoid reactions to PIABS (Fv/F0), electron transport to intersystem electron acceptors (φE0) and reduction of PSI end electron acceptors (φRo) were all substantially lower in plants grown under sub-optimal temperature than in plants grown at optimal temperature. Furthermore, thermotolerance of all the aforementioned parameters was significantly reduced by low early season growth temperatures. Among different OJIP-derived parameters, PIABS and PItotal were more heat sensitive than other fluorescence-based parameters, and φPo and φRo were the most heat tolerant. In addition, thermotolerance of PItotal (overall photosynthetic thermotolerance) was significantly correlated with thermotolerance of all other OJIP-derived parameters. Regression analysis indicated that heat tolerance trends for φE0 were more consistent with the changes in thermotolerance of PItotal. We conclude that 1) low growth temperature in the seedling phase of cotton results in decreased photosynthetic thermotolerance and 2) overall photosynthetic thermotolerance is primarily constrained by the ability of intersystem electron transport to acclimate to growth environment.