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A growing need for the development of novel hop (Humulus lupulus) varieties has emerged as a result of the increasing demand for beers with distinct organoleptic characteristics and the expected impact of climate change on hop cultivars. As the genetic variation in the existing hop cultivars is low, wild hop germplasm can be used as a source for the development of novel cultivars. In this work, we analyzed, for the first time, the genetic diversity of H. lupulus var. lupulus wild germplasm in Greece. A SNP-NGS genotyping approach using a set of nine specific genetic markers, was employed in order to determine individual genotypes and to perform population structure analyses of wild hops from a region with complex topography, namely the Region of Central Greece. Our results revealed low differentiation among populations, with the spatial genetic patterns observed relating mainly to topographical elements rather than geographic distance. Interestingly, within wild hop populations, high genetic diversity was observed, showing that in the region of Central Greece, wild H. lupulus germplasm has significant potential that can be exploited in breeding programs towards the development of local, well adapted and potentially superior hop varieties.