Abstract Maize Stalk Rot (MSR) is a serious complex disease and principally caused by Fusarium species. In order to assess pathogenicity of the Fusarium species on maize plants, a field survey was made in maize-cultivation zones in North-East China to collect the infected stem tissues from MSR. The Transmission Electron Microscopic (TEM) observation revealed thin cell wall, increased number of endoplasmic reticulum (ER), appearance of plastoglobule (PG) and presence of fungal hyphae in the MSR infected stem tissues. Further, a total of nine Fusarium strains were isolated in stems of MSR including five from Liaoning Province and four from Jilin Province. It was demonstrated that the combination of dominant strains such as Fusarium verticillioides and Fusarium graminearum displayed higher virulence than the individuals on the detached stems of seedlings. In vivo experiment showed that the MSR was obvious in maize genotype of Huangzao 4 when inoculated with the two Fusarium strains by the toothpick method. Then, the dynamic infection process observed by using eGFP-Tag strain and paraffin section revealed that F. verticillioides hyphae extended out from the cutting site to the neighboring plant cells inside the host. Thus F. verticillioides was proved to play a vital role in contribution of the MSR complex process in North-East of China.