This study presents results of electro-phoretically detectable isozyme variation in Crossing Block (CB) lines of two-rowed barley maintained by the National Crop Experiment Station. The specific objectives were to determine allelic frequencies at the four Est loci(Est1, Est2, Est4, and Est5) and their distribution over 380CB lines of two-rowed barley. A total of 17 alleles were detected over the four Est loci in these lines. There were 4 alleles (Pr, Al, Ca, and Af at the Est1 locus and their frequencies were 69.7, 1.1, 28.4, and 0.8%, respectively. At the Est2 locus, 5 different alleles (Dr, Fr, Sp, Un and a recessive null allele) were detected and their frequencies were 2.9,84.5,0.5,2.1, and 10%, respectively. four alleles (Nz, Su, At, and null were detected at the Est4 locus and the allelic frequency of Su was about 84%. Four alleles(Mi, Pi, Te, and a null allele(od)) were detected at the Est5 locus and their frequencies were 34.2, 61.0, 2.4, and 2.4%, respectively. Based on the allelic frequencies over the four Est loci, 380 CB lines were classified into 25 genotypes. The most frequent genotypes were G1(Pr-Fr-Su-Mi) and G2(Pr-Fr-Su-Pi), and their frequencies were 28.1 and 39.5%, respectively. The frequencies of other genotypes were less than 10%.