Former lignite mining areas in Eastern Germany are valuable secondary habitats for many plant and animal species endangered in the natural landscape. Here, we present a study on genetic structure and diversity of 16 populations of the threatened orchid Epipactis palustris (Orchidaceae) from five mining pits and 11 natural habitats, which we carried out in order to ascertain how many times this species immigrated into former lignite mining areas, and where the source populations are located. We used two different anonymous genetic marker methods, random amplified polymorphic DNA (RAPD) and microRNA-primed genomic fingerprinting (miRPF) to analyze patterns of genetic variation. Results of a multivariate analysis based on asymmetric Soerensen similarity, principal coordinate analysis and a neighbor-joining cluster analysis indicate high within population-variability and a moderate genetic differentiation among E. palustris populations. We found no differences between genetic diversity values of populations from former mining areas and those of natural habitats. Thus, we could not find evidences for genetic bottlenecks in the mining populations due to founder events. Source populations are predominantly close surrounding populations as geographic distance and genetic dissimilarity were correlated. However, exchanges may reach beyond 125 km and repeated independent colonization events are highly likely.