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DNA markers have potential to improve the efficiency of conventional plant breeding via marker-assisted selection (MAS). Therefore, MAS can be very effective in pistachio to eliminate undesirable progenies at early stage which can save time, cost, labor, and land. This study aimed to develop sex-linked single nucleotide polymorphism (SNP) markers for MAS, to understand sex determination system and to determine the position of sex locus in pistachio. Eight male and eight female F1 progenies from 'Siirt' × 'Bagyolu' cross, along with the parents, were subjected to RAD (restriction site-associated DNA) sequencing and 38 SNP loci were detected as putative sex-associated ones in pistachio. Validation was performed by SNaPshot minisequencing analysis in mature F1 progenies and in a wide germplasm collection. Seventeen loci successfully distinguished sex in pistachio. To ascertain cost-effective application of markers in a breeding program, high-resolution melting (HRM) analysis was performed; and 10 markers were found to perfectly separate sexes in pistachio. Because of the female heterogamety in all sex-linked SNP loci, pistachio has a ZZ/ZW sex determination system. The sex locus and sex-linked SNP markers were co-segregated in the center of the chromosome at a distance of 30.8 and 31.3 cm, respectively. This study presents sex-linked markers for MAS as well as valuable information about the sex chromosome and sex locus position in pistachio. © 2018 ISHS. Firat University Scientific Research Projects Management Unit This project was supported by the General Directorate of Agricultural Research and Policies (Project No. TAGEM/ ???ARGE/ ??I at the Republic ofe yTuMrkinistry of Food, Agriculture and Livestock, and the Çukurova University Scientific Research Projects Unit (Project No: FDK- ??? ?- ??? ?? FBA- ?? ? ?- ??? ? and FBA- ??? ?- ? ? ? ?).