The aim of the present study was to estimate genetic variation in grass sorghum (sorghum, Tunis grass, Sudan grass) and other forage species (ray grass, pearl millet, guar). Three molecular systems i.e. isozymes-PAGE, seed protein, SDS-PAGE and AFLP were used. The results indicated the presence of high similarity matrix among sorghum and ray grass; pearl millet and Sudan grass (100%) and low similarity matrix among guar and sorghum; guar and Tunis grass (44.4%) in peroxidase analysis, where the total bands was 21 with 100% polymorphism and without any unique bands. This data was different comparing with superoxidase dismutase, protein and AFLP analysis. On the other hand, the combined set data of three molecular system revealed that Tunis grass have similarity matrix in a value of (70.6%) with sorghum and (90.7%) with Sudan grass. Also, Tunis grass gave similarity matrix of (89.7%), (87.9%) and (71.2%) with ray grass, pearl millet and guar, respectively. In cluster analysis with combined data set, Tunis grass was located in sub cluster 1 with sudan grass and pearl millet in the same group, and in the same cluster of ray grass. The different clusters were found between Tunis grass, sorghum and guar. Tunis grass did not give any unique bands with all analysis but sorghum, guar and ray grass gave unique bands with protein and AFLP analysis.