Abstract: In order to clarify the pathogen structure and infection process of tobacco Fusarium root rot in Henan tobacco-growing area, the isolated pathogen from Henan tobacco-growing area were identified by morphological and molecular determinations, and the infection process of the pathogens were observed by histological staining and fluorescence microscopy. The results showed that the average detection rates of Fusarium oxysporum in southern and western Henan were 53.04% and 48.18% respectively, which were the highest; these were followed by F. solani and F. proliferatum, with average detectable rates of 33.62%, 37.24%, 6.67% and 9.91% respectively. The average detection rates of F. solani and F. oxysporum in central Henan were similar, which were 46.19% and 45.48% respectively. New pathogens such as F. commun, F. tricinctum and F. fujikuroi were detected in all tobacco-growing areas. F. oxysporum could infect the plant tissues through gaps between root epidermal cell, root hairs where root cells damage and lateral roots grow. The pathogenic spores attached to the root surface that germinated to form germ tubes which invaded the plant tissues through gaps between the plant epidermal cells and the top of root hairs, then the germ tubes grew and differentiated to form hyphae. The hyphae gradually expanded and extended along the gaps between cortical parenchyma cells, then entered the vascular bundles, eventually reproduced in large numbers in the vascular bundles and blocked the lumens of the ducts. The end result prevented water and nutrient transportation, finally caused plant diseases. Therefore, F. oxysporum, F. solani and F.proliferatum are the main pathogens of Fusarium root rot in the tobacco-growing areas of Henan. F. oxysporum can infect tobacco in a variety of ways, and this pathogen begins to infect tobacco at the early stage of contacting tobacco roots.