Abstract: To investigate the diversity of fungal community in tobacco strips during aging, the fungal ITS DNA in 12 samples of upper, middle and lower stalk leaves from four growing areas (Zhanyi in Yunnan, Changshun in Guizhou, Youxi in Fujian and Queshan in Henan) were sequenced by MiSeq. The results showed that:1) A total of 1 155 operational taxonomic units (OTUs) were obtained from the 12 samples. Samples B2F and C3F from Youxi had the highest fungal community richness and the most abundant biodiversity, respectively. 2) Ascomycota was the dominant phyla in the 12 samples. Phoma, Alternaria and Monographella were the dominant genera in samples C3F and X2F from Zhanyi, samples B2F, C23F and X2F from Queshan and sample X23F from Changshun. Aspergillus was the dominant genera in the other 6 samples. 3) 121, 79 and 168 of OTUs were common to the samples of upper, middle and lower stalk leaves from the 4 areas. The number of reads from the common OTUs accounted for more than 70% of the total number of reads in each sample, except for the sample C3F from Youxi. The differences in fungal community structure from the same stalk position of different areas mainly reflected in the proportion of dominant genera.