The new genotypes and prospective varieties with higher genetic variability are expected to be productive in a greater range of environmental conditions and thus more widely adapted, produce more stable yields when seasonal conditions vary, and offer broader protection against diseases. Evaluation and selection of parental forms for hybridization in relation to their genetic distance is essential for the breeding success. The environment can modify phenotypic expression of main traits at a different range determining yield and seed quality with respect to their quantitative inheritance. Therefore, defining the genetic distance based only on these traits is with limited value. Application of reliable laboratory methods for quick screening of a large amount of breeding materials on these indicators is necessary in this case. Present study identifies the genetic diversity of 24 genotypes with different origin- national selection, France, Portugal, Syria, Japan,etc. Varieties and lines are with different seed quality /i.e. high and low protein content/ and biotype /spring, winter and spring-winter/. Distances between groups in dendrogram arebased on: (1) biometric analysis of main traitsdetermining productive potential such as height, number of tillers, length of spikes, grain mass;(2) specific hordein profiles and (3) products of amplification with 9 ISSR primers. Field experiments and laboratory trials have been conducted in Agricultural University of Plovdiv, Bulgaria during the period 2013-2016. As a result, cluster analysis based on both genotyping and phenotyping produces two genetically distant groups of genotypes(Kamenitsa, Caravella, 82105326, 511 and 99105030 to 96105050, Kyfi, 96105027, Alexis and Orpheus). It is advisable to use pairs of parental forms from two clusters, to produce advanced germplasm in new breeding programs of two-row barley.