Abstract: Arbuscular mycorrhizal fungi (AMF) have positive effects on tobacco growth physiology, disease and stress resistance. Roots and rhizosphere soil were collected from healthy and black shank tobacco varieties K326 and Yunyan87 to investigate the effects of black shank disease on AMF diversity and community structure in roots of tobacco plants. Illumina MiSeq high-throughput sequencing technology was used to investigate the diversity of AMF in tobacco roots. The level of AMF infection in tobacco roots and the spore density in rhizosphere soil were observed by microscopic morphology. The correlation between soil physical-chemical properties and AMF infection characteristics was analyzed. High-throughput sequencing detected 1655 AMF-OTUs in tobacco roots, belonging to 1 class, 4 orders, 5 families and 6 genera. Glomus was the dominant genus of AMF in the roots of Yunyan87 healthy tobacco plants, while Paraglomus was the dominant genus in the rest samples. PLS-DA analysis showed that AMF communities in roots of healthy tobacco plants of the two varieties were relatively similar, while AMF communities in roots of black shank tobacco plants were relatively similar. The results showed that AMF infection of diseased plants decreased, and the soil spore density was also lower than that of healthy tobacco rhizosphere soil. RDA analysis showed that total potassium was the main driving factor affecting the AMF spore density and infection status of the tobacco plant, followed by available phosphorus and total phosphorus. Soil pH value was the main influencing factor of AMF diversity in tobacco roots, and there was a negative correlation between the two. In addition, potassium content in soil was the most significant factor affecting AMF community composition in tobacco roots. In this study, we investigated the difference of black shank infection on AMF in root and rhizosphere soil, and laid a foundation for further research on the relationship between AMF, soil-borne disease and plant roots.