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Hybridity and the genuineness of hybrids are prominent characteristics for quality control of seeds and thereby for varietal improvement. In the current study, the cross between two local barley genotypes (Ardhaoui: female; Testour: male) previously identified as susceptible/tolerant to salt stress in Tunisia was achieved. The hybrid genetic purity of the generated F1putative hybrids and the fingerprinting of the parents along with their offspring were assessed using a set of 17 nuclear SSR markers. Among the analyzed loci, 11 nSSR were shown polymorphic among the parents and their offspring. Based on the applied 11 polymorphic SSR loci, a total of 28 alleles were detected with an average of 2.54 alleles per locus. The locus HVM33presented the highest number of alleles. The highest polymorphism information content value was detected for the locus HVM33(0.6713) whereas the lowest PIC value (0.368) was revealed by the loci BMAC0156, EBMAC0970and BMAG0013with a mean value of 0.4619. The probabilities of identical genotypes PI for the 11 microsatellite markers were 8.63 × 10−7. Banding patterns among parents and hybrids showed polymorphic fragments. The 11 SSR loci had produced unique fingerprints for each analyzed genotype and segregate between the two parental lines and their four hybrids. Parentage analysis confirms the hybrid purity of the four analyzed genotypes. Six Tunisian barley accessions were used as an outgroup in the multivariate analysis to confirm the efficiency of the employed 11 nSSR markers in genetic differentiation among various barley germplasms. Thus, neighbor joining and factorial analysis revealed clearly the discrimination among the parental lines, the four hybrids and the outgroup accessions. Out of the detected polymorphic 11 nuclear SSR markers, a set of five markers (HVM33, WMC1E8, BMAC0154, BMAC0040and BMAG0007) were shown to be sufficient and informative enough to discriminate among the six genotypes representing the two parental lines and the four hybrids from each others. These five nSSR markers presented the highest number of alleles per locus (An), expected heterozygosity (He), PIC values and the lowest probabilities of identity (PI). These nSSR loci may be used as referral SSR markers for unambiguous discrimination and genetic purity assessment in barley breeding programs.