Minuartia smejkalii is an obligate serpentinophyte plant endemic to the Czech Republic. Since the 1960s, the species’ habitat has undergone strong human-mediated fragmentation, resulting in extinction of some populations and dramatic size reduction of the remaining populations. Thus, contrary to the typically stable serpentine habitats, M. smejkalii habitats underwent a recent and severe decline, which can exacerbate the effects of fragmentation on population genetic structure. We examined the genetic structure of all known M. smejkalii populations and two populations of M. corcontica and M. caespitosa, which are closely related, using RADSeq. The results indicate low, but clear differentiation among the three species, thus supporting the status of M. smejkalii as an independent taxon, though more extensive analysis of the whole group is needed. We further show high genetic diversity within M. smejkalii populations, low to moderate among-populations differentiation, and moderate regional differentiation. This could be due to the outcrossing mating system of M. smejkalii promoting high levels of gene flow and historical factors (multiple founder events, a recent bottleneck and/or a genetic time lag). We finally demonstrate that 2–3% of the markers show differentiation patterns consistent with divergent selection, suggesting that some local adaptation might have occured in M. smejkalii. Based on our observations, but without any experimental testing for local adaptation, if a conservation action is to be carried out, we recommend strictly separating the material from the two regions, and if possible, separating the populations within a region.