The effects of glycine betaine (GB) under heat stress conditions were studied in three marigold cultivars, namely, ‘Narai Yellow’, ‘Bali Gold’, and ‘Columbus Orange’. GB was foliarly applied to the seedlings 24 hours before transfer to either 25°C/25°C or 39°C/29°C (day/night) conditions for 15 days. Heat stress conditions caused photoinhibition and low levels of CO₂ assimilation rate (A), stomatal conductance (g_s), and transpiration rate (E) in all marigold cultivars compared with those of control plants grown under 25°C/25°C conditions. The accumulation of reactive oxygen species (ROS), lipid peroxidation, and cell death were also higher under heat stress in all cultivars compared with those in the control. However, the effect of heat stress on relative water content (RWC) was statistically significant only in ‘Bali Gold’. Foliar applications of GB at 0.5 and 1 mM alleviated photoinhibition and resulted in higher A, g_s, and E in all marigold cultivars compared with those in the control under heat stress. Application of GB also resulted in lower levels of hydrogen peroxide, superoxide, lipid peroxidation, and cell death in all cultivars. The effect of GB on improving RWC was significant only in ‘Bali Gold’. In most cases, there were no significant differences between the effects of GB at 0.5 and 1 mM. The effect of GB seems to be very consistent across all marigold cultivars, as suggested by the lack of interaction between this effect and cultivars in most of the parameters that were studied. Overall, these results indicate that the foliar application of GB could possibly be used to mitigate the effect of heat stress in marigold.