Introduction Walnut (Juglans regia L., 2n = 32, ~ 606 Mb per 1 C genome) is a monoecious, dichogamous, wind-pollinated, long-lived, perennial tree species belonging to the family Juglandaceae (Zhu et al., 2015). After cashew (Anacardium occidentale L.) and almond [Prunus dulcis (Mill.) D. A. Webb], walnut is the third most widely planted nut tree worldwide with a global annual production (with shell) of around 2.2 × 10 6 metric tons 1. Turkey is the fourth largest walnut-producing country in the world after China, USA, and Iran 1. The kernel of walnut has a high economic impact on the food industry and is very popular and valued around the world due to its health-related, sensorial, and nutritional properties (Martinez et al., 2010). Many researchers have demonstrated the beneficial health effects of human diet supplemented by walnut due to its high protein, oil vitamin and mineral content (Dzhafarova et al., 2009; Martinez et al., 2010). Recent research also suggests that in addition to nuts, the extracts of walnut leaves have a positive impact on lipid metabolism and decrease blood sugar levels in human beings (Dzhafarova et al., 2009). On the other hand, leaves, shells, green walnuts, kernels, and bark of walnut tree have been used in pharmaceutical and cosmetic industries. Fresh leaves of walnut are utilized for the treatment of skin inflammations, ulcers, and hyperhidrosis in the pharmaceutical industry and for their antiseptic, anthelmintic, astringent, and antidiarrheal properties in folk medicine, and dry walnut leaves are used as infusions. Additionally, green husk of walnut is the basic material for the conventional liqueur of walnut, and shells are used as a filtration medium for the separation of crude oil from water. As a result of these great health benefits and commercial importance of walnut, detection of its genetic diversity and relationships among accessions has become significant topics in studies on germplasm conservation and plant breeding (Pereira et al., 2007). In Turkey, nongrafted walnut seedlings had been used in new walnut plantations until the beginning of 1970s and commercial walnut orchards were established with