In order to investigate the changes of genetic integrity and stability of tobacco seeds after a long-term storage and update, several tobacco cultivars’ botanical characteristics and agronomic traits were tested. The results showed that botanical characteristics, including plant style and leaf style, changed in some degree after 20-year storage and seed update, which might be attributed to genetic variation. Some agronomic traits had significant change, such as plant height, leaf number, stem width, which were mainly caused by the environmental condition.