The variation among some barley germplasm (Giza 123, Giza 127, Giza 130, Desert, and Sinai) was investigated comparatively at the molecular and biochemical levels. Leaf DNAs extracted from different barley germplasm were amplified with randomly chosen primers in a Polymerase Chain Reaction (PCR) using Random Amplified Polymorphic DNA (RAPD) technique. Only five primers amplified all DNA templates among 15 primers. Primers generated a total of 112 DNA fragments ranged from 1.2 to 0.1 kbp. Out of the 112 fragments, 16 (14.29%) were polymorphic. The result indicated that fragments generated per primer, with an average of 22.4 products, where the number of polymorphic bands per primer, with an average of 3.2 polymorphic bands. In addition, the genetic variation in the anti-nutritional factors and nutrient composition of the same genotypes were determined. A wide variation in total phenolic, phytic, tannin, vicine, and saponin contents was found among these germplasm. The range of total phenolic was 3.83 to $7.50;mg;g^{-1}d.w$. Among all germplasm, Desert germplasm was characterized by the highest tannin content. However, saponin ranged between $5.66;mg;g^{-1}d.w$. (in Giza 123) to $9.21;mg;g^{-1}d.w$ (in Sinai) germplasm. The concentrations of the phytic acid were generally low, ranging between 0.99 to $6.78;mg;g^{-1}d.w$. The germplasm Desert and Sinai had the highest level of vicine, 1.89 and $1.87;mg;g^{-1}d.w$., respectively. The results of the molecular characterization and anti-nutritional content can be used as the starting point needed to identify the valuable Egyptian barley germplasm.