The Present study was carried out to imploy the polyacry lamide gel electrophoresis (SDS–PAGE) characterization of Serw 1, as a parent, Gemmiza 1 as a mother varieties and their first generations polycrosses a long 50,100, 150 and 200 meter distances from the parent variety (Serw 1). To determine isolation distance (50m) between cultivated Egyptian clover, polyacrylamide gel electrophoresis (SDS – PAGE) was employed to detect variation in total soluble protein content technique among five distances of Egyptian clover namely, 200m 150, 100, 50m and Gemmiza 1 distance. After detection of isolation distance with 50m, different protein fragmentation was recorded for the cultivars which ranged from sixteen to eleven for distance 100, 200m and Gemmiza 1; respectively, with molecular weight ranged from 207 to 46 KDa. Finding of similarity and dissimilarity for protein patterns of cultivated Egyptian clover showed that, distances No. Serw 1 and 50m showed same protein patterns with almost protein loci and molecular weights with fourteen protein bands. According to electrophoretic study, phyllogenetic tree was constructed and indicated clear genetic base for distances Serw 1 and 50m which belongs to the same subgroup. Our previous findings detected isolation distance with 50m could cause genetical mixing probability between Serw 1 and 50m cultivars which represented as male and females; respectively. A chart of minimum recommended isolation distances for Egyptian clover, the chart includes three minimum distances recommendations: - Isolation distance of 200m for foundation seed. - Isolation distance of 150m for registered seed. - Isolation distance of 100m for certified seed.