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Prunus L. (Rosaceae), a genus of over 230 species, exhibits gametophytic self-incompatibility system (GSI). In this study, we explored the patterns of variability in Plum genotypes carrying different reproductive systems, using 36 microsatellite markers (SSRs) and the S-locus. The analysis of genetic diversity showed a rich genetic variability of the studied genotypes. Linkage Disequilibrium decayed rapidly in analyzed plums reaching the significant value (r² = 0.1) at 7.5 cM. Also, we examined the patterns of variability at the end of the sixth linkage group to emphasize the effect of the S-locus on the surrounding region. The genetic diversity in the genomic neighborhood of the S-locus is related to the negative frequency dependent selection (NFDS) which can extend the high polymorphism at the selected locus to closely linked neutral sites. We conducted a drought stress on Tunisian plum to test their response. The analysis of five morphological parameters tested under drought conditions allowed selecting five plum genotypes (one hexploid and four diploid) characterized by their important tolerance to drought stress. These genotypes may be selected as important rootstock for varietal improvement.