The aim is analyzing genetic reLationship and identifying varieties by detecting DNA differences of three kinds of Nicotiana tabacum L. using Fourier infrared spectrum (FTIR). Results show that DNA FTIR of three kinds of Nicotiana tabacum L. is relatively similar. They all have four obvious characteristic peaks. 1 105 cm-1 beLongs to symmetrical stretching vibration of phosphodiester bond, 1 250 cm-1 is unsymmetrical stretching vibration of phosphodiester bond, 1 400 cm-1 is contributed to glucosidic bond, and 1 622 cm-1 belongs to C4C5C6 stretching vibration of cytosine. DNA FTIR data was handled by smoothing, standardizing, second derivative, principal component analysis and Hierarchical cluster analysis. The standard model of Hierarchical cluster combined with principal component of the second derivative was set up. The correct rate of identification is 100%. Yunyan 87 and K326 were clustered into one by using the model. The distance coefficient is 0.003, and DNA similarity is 99.7%, Hongda was clustered into one by itself. The correct rate of cluster is 100%. The study provides a reference for Nicotiana tabacum L. variety identification and genetic breeding.