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Phoenix dactylifera L. were analyzed for DNA sequence diversity in the non-coding region of chloroplast DNA. The trnL-trriF intergenic spacer sequences were used as genetic markers to establish refined genetic relationships for differentiating date-palm accessions collected from Tunisian oasis. A single DNA band of approximately 400 bp was amplified from each Tunisian date-palm cultivars. Using the Kimura-2 method, nucleotide sequences were aligned using the MEGA 5.2 program to calculate pairwise divergence of trnL-trnF spacer sequences between cultivars. The relatively high AT values (60.5-67.6%) of trriL-trriF intergenic spacer may explain the high proportion of the identified transversions (ti/tv = 0.67). These results suggest the rate of nucleotide diversity with a great variation level of chloroplast non-coding region. The analyzed data illustrated a considerable level of variability in the genetic pool of the local germplasm. In fact, relationships inferred from the cpDNA analysis suggest several clades, which do not show geographical correspondence. In the other hand, 14 haplotypes were detected among 31 individuals examined, yielding a haplotype diversity of 0.854 and a level of nucleotide diversity of (0.0081). Selective neutrality tests show that Tajima’s as well as Fu and Li’s tests were negative and not significant in the studied cultivars. The cytoplasm variability indicates a narrow genetic base within date-palm cultivars. The opportunity of the designed methods is discussed with the molecular characterization of genotypes in order to enhance the conservation and the improvement of the local date-palm germplasm. (C) 2014 Elsevier B.V. All rights reserved.