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Lavender species are widely distributed in their wild forms around the Mediterranean Basin and they are also cultivated worldwide as improved and registered clonal varieties. The economic interest of the species belonging to the Lavandula genus is determined by their use as ornamental plants and important source of essential oils that are destinated to the production of cosmetics, pharmaceuticals and foodstuffs. Because of the increasing number of cases of illegal commercialization of selected varieties, the protection of plant breeders' rights has become of main relevance for the recognition of breeding companies' royalties. With this aim, genomic tools based on molecular markers have been demonstrated to be very reliable and transferable among laboratories, and also much more informative than morphological descriptors. With the rising of the next-generation sequencing (NGS) technologies, several genotyping-by-sequencing approaches are now available. This study deals with a deep characterization of 15 varietal clones, belonging to two distinct Lavandula species, by means of restriction-site associated DNA sequencing (RAD-Seq). We demonstrated that this technology screens single nucleotide variants that enable to assess the genetic identity of individual accessions, to reconstruct genetic relationships among related breeding lines, to group them into genetically distinguishable main subclusters, and to assign their molecular lineages to distinct ancestors. Moreover, a number of polymorphic sites were identified within genes putatively involved in biosynthetic pathways related to both tissue pigmentation and terpene production, useful for breeding and/or protecting newly registered varieties. Overall, the results highlighted the presence of pure ancestries and interspecific hybrids for the analyzed Lavandula species, and demonstrated that RAD-Seq analysis is very informative and highly reliable for characterizing Lavandula clones and managing plant variety protection. [ABSTRACT FROM AUTHOR]