Abstract: In order to explore the molecular genetic basis of proline heterosis in tobacco under drought stress, 7 tobacco varieties with different drought resistance and 10 hybrid combinations were used as materials, and drought stress treatments were set up to analyze proline heterosis and differential expression of related genes in tobacco by pot experiments.The results showed that the content of tobacco proline under drought stress increased continuously at the early stage and decreased after the 14th day.The heterosis of proline content in hybrid combinations was significantly different, and the proline content had the mid-parent heterosis with the highest value of 40.35 and the lowest of-51.66.The relative expression levels of the proline synthesis key enzyme genes P5CS and δ-OAT were significantly higher in the strong-dominant combinations, being 2.10 times and 1.87 times of those of the weak-dominant combinations, respectively.There was a significant positive correlation between the proline heterosis and P5CS gene relative expression after persistent drought for 7d at the vigorous growth stage.The correlations between the proline heterosis and δ-OAT gene relative expression were significant or extremely significant after persistent drought for 14d at the root extension stage and persistent drought for 7d and 14d at the vigorous growth, with correlation coefficients of 0.93, 0.98 and-0.96, respectively.Therefore, the up-regulation or down-regulation of δ-OAT and P5CS was the molecular basis for the formation of heterosis of tobacco proline content during the corresponding growth periods.