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Characterizing genetic diversity and structure of populations is essential for the effective conservation of threatened species.Orchis patenssensu lato is a narrowly distributed tetraploid species with a disjunct distribution (i.e., Northern Italy, North Africa and the Canary Islands), which is facing a severe decline. In this study, we evaluated levels of genetic diversity and population structuring using 12 new nuclear microsatellite markers. Our analyses of genetic differentiation based on multiple approaches (Structure analysis, PCA analysis, andF-statistics using the ploidy-independentRho-index) showed that gene flow is low across the range ofO. patens s.l., particularly in the Canary Islands. Clear differences in allele frequencies between Italy, Algeria and the Canary Islands underlie the genetic differentiation retrieved. Our study provides support for the recognition ofO. canariensisas a sister species toO. patensand the separation of the Italian populations as a new subspecies ofO. patens. Despite the high heterozygosity values found in all populations (ranging from 0.4 to 0.7), compatible with the tetraploid status of the species, small population sizes and reduced gene flow will be likely detrimental for the different populations in the long term, and we recommend immediate conservation actions to counteract further fragmentation and population decline.