Abstract: To understand the diversity and community structure of fungi on Yunnan flue-cured tobacco leaves during aging at different locations, and to explore beneficial microbes in aging process, the diversity and community structure of culturable fungi of flue-cured tobacco leaves during aging from different locations were studied by 18S rDNA coloning and sequencing technology. The results showed that the number of fungi on the surface of tobacco leaves gradually reduced with the aging time, except that of Qujing in June. It was found that the numbers of fungi on the surface of tobacco during aging in Qujing and Chuxiong were very close, and the amount of fungi on aging tobacco leaves in Yuanjiang was the least. Based on morphological characteristics and 18S rDNA sequencing analysis, twenty four fungal genera (species) were identified, such as Rhizopus spp., Penicillium spp., and Aspergillus spp., etc. The number, diversity and community structure of fungi on the surface of flue-cured tobacco leaves varied with aging locations and periods, however, Rhizopus spp. was always the predominant genus.