Sesame Fusarium wilt (SFW), which is caused by Fusarium oxysporum f. sp. sesami (FOS), is one of the main fungi diseases and primarily occurs at vegetative growth and flowering stages in sesame. In order to accurately evaluate the resistance level of sesame germplasm accessions to FOS strains during vegetative stage (from two-pair-leaves period to budding period), we analyzed the Fusarium wilt symptoms and the disease index (DI) variation of sesame germplasm accessions treated with several FOS strains, various concentrations of microconidia suspension and different time with treatment. A precise evaluation method of sesame resistance to Fusarium wilt disease during vegetative stage was established. Results indicated that Fusarium wilt symptom occurred in sesame plantlets 1 to 2 weeks later after inoculation with 1 × 106 microconidia/ mL FOS suspension. The DI values became stabilized after 4 weeks. The resistance level of germplasm accessions and the pathogenicity of FOS strains were reflected using the above established method. Five grades of Fusarium wilt resistance from grade 0 to 4 for sesame during vegetative stage was proposed. The results derived from tests of 42 sesame accessions revealed that the wild species, S. radiatum exhibited the stable high resistance to FOS (DI = 0), while S. angustifolium was highly susceptible (DI = 100). Of the 40 cultivated accessions, 57.5% samples were highlysusceptible (HS), while 27.5% were showing high- or medium-resistant. The findings supply the technological aids in further analyzing the genetic mechanism of sesame resistance to Fusarium wilt disease.