Primary hexaploid tritipyrum lines are amphiploids between Triticum durum and Thinopyrum bessarabicum, which can set seed in at least 250 mM NaCl. Genetic variation within and among 14 lines of primary tritipyrum were investigated by using 2 random and 6 semi random primers that produced clear and reproducible DNA amplicons. Average values for Nei's gene diversity (h) and Shannon indices (I) were 0.115 and 0.178, respectively. These results indicated low variation within primary tritipyrum lines, probably related to unbiased chromosome and partial aneuploidy. Total genetic diversity (Ht) and gene diversity within lines (Hs) were 0.2114 and 0.1156, respectively. The coefficient of genetic differentiation (Gst) was equal to 0.4534. AMOVA showed highly significant (P<0.001) genetic differences among lines. From the total genetic diversity, 51.54% is attributable to 14 tritipyrum lines and 48.16% to differences within lines. Fixation index (FST) was 0.4815. Genetic distance, calculated on the basis of allele frequencies among lines, ranged from 0.064 to 0.226 with an average of 0.1156. Grouping of populations based on UPGMA and Nei's gene diversity are indications of genetic similarity between some populations. Pop6 and populations located in group III had the highest genetic distance. These results could be used in breeding and cross programs to breed a new tritipyrum plant.