Abstract: With six different genotype flue-cured tobacco varieties as the materials，genotype difference of flue-cured tobacco seedling were studied with the main root development, the lateral root development, biomass, roots activity and chlorophyll content by indoor culture and floating system. The results indicated that the main root development was statistically different in 96h or 144h tobacco seeding, however that was not was statistically different in 192h or 240h tobacco seeding. The number and total length of different genotype tobacco’s first, second, third and forth order lateral root was increasing with the increasing seedling age. The second lateral root, which total length was higher than any of the first, the third and the forth lateral root, was the main absorbing nutrient roots of tobacco seedling. The biomass from different tobacco seedling was different. The roots activity was different obviously and the chlorophyll of 5th leaf at the age of 50d seedling was not different statisticly. The cholorophyll content of Hongda was highest but that of NC82 was lowest of all tested variety.