The inter-simple sequence repeat (ISSR) markers have been used in order to determine genetic variation and relationship between two clover ecotypes. Ten (10) primers for ISSR were used in this study but only six were successful in generating reproducible and reliable amplicons for different types of the Egyptian clover. The results reveal the polymorphism level by ISSR primers. HB10 ISSR-primer was higher than the rest of the ISSR primers in polymorphic 100%. The Fahl monocut ecotype had 29 present bands, 3 absent bands in total of 32 bands; among those there were two unique bands. The multicut ecotype were given different pattern of bands, Gemmiza1 (21 present and 11 absent), Giza6 (21 present and 11 absent) and Serw1; (23 present and 9 absent). There were three unique bands appearance in the two ecotypes. Fahl was given two with HB11 and HB13; the Serw1 multicut cultivar had one unique bands with HB08. Similarity indices among the four Egyptian clover cultivars based on ISSR analysis was estimated and the highest value appeared between Fahl and Gemmiza1 as well as Giza6 and Serw1 followed by Fahl and Serw1. The lowest similarity value appeared between Gemmiza1 and Serw1 followed by Gemmiza1 and Giza6.