In order to assess the genetic variability among regenerants. field trials were conducted with 64 barley lines derived from tissue culture (TCD lines) of four Bulgarian barley varieties. The results indicate that the agronomic performance was altered by tissue culture regeneration and the frequency of variation was cultivar dependent. Seven TCD lines showing desirable agronomic characters were selected in the SC16 progeny. Molecular markers (protein, restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) have used applied to determine the variability induced by tissue culture. Polymorphism was detected in sequences coding for C‐hordeins in line 70 derived from cv.‘Jubiley’Two lines from cv.‘Ruen’exhibited polymorphic bands after hybridization with a mitochondrial DNA probe. RAPD assays have been carried out using 20 different l0‐mer primers. Heritable polymorphisms in several TCD lines have been observed.