Abstract: Tobacco brown spot is a fungal disease occurring at the late stage of tobacco maturation, which is mainly harmful to tobacco leaf and seriously affects tobacco production in China. In order to understand the carbon and nitrogen source utilization phenotypes of Alternaria alternata and Alternaria longipes which are two main pathogenic fungi of tobacco in China. CQ1 (moderately virulent) and GZ11 (moderately virulent) of A. alternata as well as HuN2 (most virulent) and YN4 (weak virulent) of A. longipes were selected to analyze the utilization of 190 species of carbon source materials in the microplates of PM1 and PM2, 95 species of nitrogen source of PM3 using the Phenotype Microarray System. The results revealed that the utilization of carbon and nitrogen sources of the four tested strains were roughly similar, the utilization ratio of carbon and nitrogen sources were 24.21% and 86.31%, respectively. There were also some differences among them, for A. longipes, the most virulent HuN2 had a better use of D-Mannose, D-Xylose, D-Mannitol, b-Cyclodextrin, L-Valine, D-Asparagine, L-Asparagin, Adenosine, i-Erythritol, and so on than the weak virulent YN4. For A. alternata, the moderately virulent CQ1 had a better ability in using L-Rhamnose, Laminarin and Dihydroxyacetone than GZ11. Additionally, the weak pathogenic strain YN4 was better than HuN2, CQ1 and GZ11 in using Salicin which indicated that there were obvious utilization variance in different types of pathogenicity.