In this study, we aimed to develop novel genic simple sequence repeat (eSSR) markers and to study phylogenetic relationship amongPistaciaspecies. Transcriptome sequencing was performed in different tissues of Siirt and Atl cultivars of pistachio (Pistacia vera). A total of 37.5-Gb data were used in the assembly. The number of total contigs and unigenes was calculated as 98,831, and the length of N50 was 1,333 bp after assembly. A total of 14,308 dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide SSR motifs (4-17) were detected, and the most abundant SSR repeat types were trinucleotide (29.54%), dinucleotide (24.06%), hexanucleotide (20.67%), pentanucleotide (18.88%), and tetranucleotide (6.85%), respectively. Overall 250 primer pairs were designed randomly and tested in eightPistaciaspecies for amplification. Of them, 233 were generated polymerase chain reaction products in at least one of thePistaciaspecies. A total of 55 primer pairs that had amplifications in all testedPistaciaspecies were used to characterize 11P. veracultivars and 78 wildPistaciagenotypes belonging to ninePistaciaspecies (P. khinjuk,P. eurycarpa,P. atlantica,P. mutica,P. integerrima,P. chinensis,P. terebinthus,P. palaestina, andP. lentiscus). A total of 434 alleles were generated from 55 polymorphic eSSR loci with an average of 7.89 alleles per locus. The mean number of effective allele was 3.40 per locus. Polymorphism information content was 0.61, whereas observed (Ho) and expected heterozygosity (He) values were 0.39 and 0.65, respectively. UPGMA (unweighted pair-group method with arithmetic averages) and STRUCTURE analysis divided 89Pistaciagenotypes into seven populations. The closest species toP. verawasP. khinjuk.P. eurycarpawas closerP. atlanticathanP. khinjuk.P. atlantica-P. muticaandP. terebinthus-P. palaestinapairs of species were not clearly separated from each other, and they were suggested as the same species. The present study demonstrated that eSSR markers can be used in the characterization and phylogenetic analysis ofPistaciaspecies and cultivars, as well as genetic linkage mapping and QTL (quantitative trait locus) analysis.