The efficacy of genetic material for use as DNA barcodes is under constant evaluation and improvement as new barcodes offering better resolution and efficiency of amplification for specific species groups are identified. In this study, the chloroplast intergenic spacer psbK-psbI was evaluated for the first time as a DNA barcode for distinguishing date palm cultivars. Nucleotide sequences were aligned using MEGA 6.0 to calculate pairwise divergence among the cultivars. The analyzed data illustrated a considerable level of variability in the genetic pool of the selected cultivars (0.009). In fact, five haplotypes were detected among 30 cultivars examined, yielding a haplotype diversity of 0.685. An unweighted pair group method with arithmetic mean phylogenetic tree was constructed and shows a well-defined relationship among date palm cultivar varieties. On the other hand, selective neutrality investigations using Tajima test and Fu and Li tests were negative, providing evidence that date palm has been undergoing rapid expansion and recent population growth. Thus, we suggest that the psbK-psbI spacer can be successfully used to construct reliable phylogenetic trees for P. dactylifera.