The genetic diversity of major characteristics in 302 flue-cured tobacco germplasm from Guizhou Tobacco Research Institute was analyzed and compared in this paper for their further utilization. The results showed that 4 morphological characters, 8 agronomic characters, 7 chemical constituents were wildly variant. The indexes of genetic diversity (H’) were relatively high on plant height, stalk circumference, pitch, leaf length, leaf width and growth duration after transplant. However, the H’ of nicotine, total sugar, total nitrogen, potassium and protein were highly aberrant. For the smoking results, the H’ of aroma quality, flavor, offensive odor, total score were aberrant, and the H’ among morphological characters were also abundant.