Agonoscena pistaciae Burckhardt & Lauterer is a serious economic pest of pistachio in the world. Despite its economic importance, little is understood of its genetic diversity and population relatedness. We used two mitochondrial genes (COI and Cyt-b) and one nuclear (ITS2) sequence to investigate genetic structure and diversity of 5 populations spanning the distribution of this pest in Iran. High levels of genetic diversities were found for all three genes due to the existent of two genetically divergent lineages. Haplotype network and phylogenetic analyses separated populations into two major clades, the central – southeastern and the northeastern clades. Because these lineages are genetically highly different with the genetic divergent of 4.57% – 4.99% and 3.61%-4.58% based on cytochrome c oxidase I and Cyt-b and 2.88% - 4.91% for nuclear 5.8S/ITS2 sequences suggesting possibility of existent of cryptic species of A. pistaciae in Iran. Genetic structure analysis revealed that geographic adjacent populations share haplotypes while distant populations exhibit distinct haplotypes, indicating geographic isolation limited gene flow among populations. This finding is consistent with population pairwise FST analysis which found that most populations were genetically distinct. The high level of population genetic structuring is might probably related to dispersal capacity, life history variation and the geographic isolation among the populations.

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