Diversity in Pistacia has been evaluated at all molecular levels using the internal transcribed spacer 1 (ITS1) marker in three species (Pistacia atlantica subsp. atlantica; Pistacia vera and Pistacia terebinthus), and compared with other Pistacia species. Results showed that the ITS amplification and sequencing, followed by phylogenetic analyses, identify the species and confirm their classification, which revealed that it can be used as a marker. Our results suggest that ITS1 analyses might provide a simple and inexpensive approach to validate the species of samples collected from the natural population, where species identification can be difficult, especially if hybrids are present or if the season is not optimal for identifying differences in morphological traits.