Abstract: In order to investigate the diversity and community structure difference of arbuscular mycorrhizal fungi(AMF)in roots of four flue-cured tobacco varieties(Yunyan 202, Yunyan 87, Yunyan 85 and K326), the 18S rRNA gene clone libraries of AMF were constructed. The results showed that there were 21 operational taxonomic units(OTUs)in the four clone libraries. The number of OTUs in Yunyan 202, Yunyan 87, Yunyan 85 and K326 clone libraries were 6, 9, 8 and 8, respectively. According to the distribution of OUTs in the four tobacco varieties, OTU1 and OTU2 were the main types, with OTU3 being a common type and OTU4 only found in Yunyan 85 and K326. The other remaining gene types were only appeared in 1 of the four tobacco varieties. Phylogenetic analysis showed that 9 OTUs could be classified into genus:5 of the OTUs belonging to Glomus, 2 of the OTUs belonging to Acaulospora and another 2 OTUs belonging to Sclerocystis. The taxonomic of the other 12 OTUs was not clear, they might belong to novel AMFs. Diversity analysis suggested that the AMF diversity of Yunyan 87 was the most abundant and there was no obvious difference between Yunyan 85 and K326. The type of AMF in Yunyan 202 was the least. In summary the main AMF types in the four tobacco varieties were the same, while rare AMF types were significantly different.