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The aim of this paper was to determine how the pistachio trees evolve, and the evolving factors that influence pistachio population establishment. For that, we investigated pistachio genetic structure from some regions of Tunisia, by sequencing two noncoding chloroplastic regions (trnL (UAA) and trnL-trnF intergenic spacer). We found strong genetic diversity among groups, with the absence of high differentiation between population pairs. A deep phylogeographical break separated two major clusters: "El-Guetar and "Gafsa/Sidi-Bouzid. This conclusion is proved by the haplotype networks, the phylogenetic trees, and the molecular variance analysis. Different interpretations were proposed to explain this cytoplasm dimorphism, based on the molecular evolution and demographic history analysis: (1) the domestication events, which are very important to understand the variability between the prospective areas, (2) the gene flow between them, a process that occurs both in time and in space through pollen and seeds, and strongly interacts with the local farming systems, (3) the geographical barriers that exist, which limit gene flow transfer and make particular climatic conditions of the El-Guetar oasis. Given that, the genetic diversity study within Tunisian pistachio cultivars is very useful to contribute to the national management effort for the improvement and conservation of pistachio genetic resources. Moreover, in 2017, Tunisia ranked among the top 10 countries in global pistachio production. Thus, studying the diversity of Tunisian pistachio can make an important impact on global production.