Abstract: Background and Objective: Sesame diseases, which are mostly caused by fungal pathogens, are important production restrictions in Egypt and climatic change is exacerbating their impact. The objectives of this study were to identify high yield lines, to find out the phytochemical components in the ethanol extracts of genotypes and to determine some markers related to genes controlling the Fusarium wilt resistance trait. Materials and Methods: Four elite sesame lines and commercial variety Shandaweel were evaluated based on seed yield ha–1 over two seasons. And they tested for seed-borne fungi. Then these genotypes were examined using R-ISSR. Results: Significant variations (p<0.05) found among genotypes for seed yield ha–1. The highest yield was obtained from Shandaweel. The line C3.8 recorded the low fungal incidence and achieved the highest germination and its seed has chemical compositions that have antifungal activity and absent in Shandaweel. R-ISSR generated more markers linked to Fusarium wilt resistance compared with RAPD and ISSR when used alone. Line C3.8 contains defense response genes against fungi diseases, which produced bioactive chemical compounds that have conferred resistance. Conclusion: Line C3.8 will be used as a parent in other breeding programs to form new varieties resistant to Fusarium wilt in sesame. And, this line can use as a basis for generating new broad-spectrum antimicrobial formulations.