Abstract: Two tobacco cultivars K326 and Xiangyan 4 were selected to investigate nutrient deficiency symptoms, root morphology, and biomass under the nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), sulfur (S) and iron (Fe) deficient treatments in order to identify the effects of nutrient deficiencies on tobacco seedling. The results indicated that the shoots and roots of both cultivars appeared specific symptoms under 7 element deficiency treatments. Among them, Fe deficiency resulted in the appearance of the earliest deficient-symptom, but S deficiency resulted in the latest symptom. Compared to the control, N deficiency promoted root growth of K326 and Xiangyan 4, and the root biomass increased by 80.0% and 78.1%, respectively. However, N deficiency decreased shoot biomass of both tobacco cultivars. S deficiency had the least effect on root morphology and plant biomass of two cultivars. Under the other element deficiency treatments, the biomass of shoots and roots decreased remarkably for both cultivars. The lateral root number of K326 also declined rapidly, however, for Xiangyan 4, its lateral root number reduced sharply only under Mg and Fe deficient treatments.