Fusarium oxysporum f. sp. sesami (Fos) is an extremely destructive pathogen, causing sesame Fusarium wilt disease worldwide. To clarify the pathogenicity and the genetic characters of Fos, we systematically investigated 69 F. oxysporum isolates collected from major sesame-growing areas in China. Among these isolates, 54 isolates were pathogenic and 15 were non-pathogenic according to pathogenicity testing on sesame seedlings. For the pathogenic isolates, three Fos pathogenic groups were defined based on the three differential sesame hosts for the first time. A translation elongation factor 1a (EF-1a) gene tree was constructed to determine the genetic diversity of the F. oxysporum isolates, but could not separated Fos isolates from the non-pathogenic isolates and other F. oxysporum formae speciales. Ten Secreted In Xylem (SIX) genes, one family of effectors, were identified in Fos isolates by a search with the genome data, and were subsequently screened in the 69 F. oxysporum isolates. Compared with the SIX gene profiles in other F. oxysporum formae speciales, the presence and sequence variations of the SIX gene homologues directly correlated with the specific pathogenicity of Fos toward sesame. Furthermore, eight of these Fos SIX genes were significantly expressed in sesame plants as infection of the Fos isolate. The findings have important significance for understanding the pathogenic basis of Fos isolates, and will contribute to improve the diagnostics to effectively control Fusarium wilt disease in sesame.