The shape information of leaves from 39 tobacco varieties was extracted by using landmark method. The differences in leaf shapes were compared and analyzed among different varieties and different leaf positions at different growth stages. Principal component analysis was used to reduce the dimensionality of the data. The sources of differences were visualized among different leaf shapes. Decision tree, random forest and support vector machine were used to perform discriminant analysis on tobacco leaf shapes. The results of the principal component analysis showed that the first three principal components accounted for 42.7%, 21.3% and 10.7% of the total differences in tobacco leaves at the flowering stage, which were characterized by leaf width and the maximum width position, leaf torsion, and petiole size, respectively. The discriminant results of tobacco leaf shape based on machine learning showed that the discriminant accuracy based on landmark data was 52%-62%, while the value was 51%-54% for common leaf shape indicators. The discriminant accuracy on superior or medial leaves was about 10% higher than that of inferior leaves, representing more obvious characteristics of variety. Due to the growth of the leaves, the discriminant accuracy of the leaves at rosette stage was nearly 10% lower than flowering stage. The discriminant accuracy of landmark method increased to 77% after removing 12 atypical varieties. The effect of the landmark method on leaf shape information extraction is better than the common leaf shape indicators, which provides a new idea for the automated extraction of leaf shape information.