We examined the genetic diversity and structure in populations of the endangered grassland herb Vincetoxicum atratum using 11 polymorphic microsatellite loci. Although the populations were small and disconnected, our molecular data indicated that the species maintains relatively high levels of genetic diversity and connectivity among populations. Population clustering analyses detected two to three clusters and most of the populations of V. atratum comprised admixture of these genetic clusters. These admixtures likely formed during the process of colonizing habitats that had been disturbed by human activities. However, STRUCTURE clustering detected low-admixtures in populations occurring in rocky maritime sites, which may not be suitable for agriculture/rangeland activities. High genetic diversity and population connectivity suggested that loss of the remaining populations by grassland reduction might be an immediate threat for this species. Small grasslands populations managed by local farmers need appropriate conservation practices. Although our results showed genetic diversity and gene flow among populations of V. atratum were high, it is possible that this resulted from the historical continuous distribution of the species. To examine this hypothesis, further periodical monitoring of the genetic diversity and the genetic differentiation for the species is needed for a conservation action of the species.