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High Andean wetlands are naturally fragmented ecosystems that are impacted by anthropogenic activities. Although they constitute important reservoirs of biodiversity and ecosystem service providers, many aspects of their ecology are still unknown. In this study, we investigated the population genetic structure of two dominant and highly interactive plant species of high altitude wetlands, Patosia clandestina (Juncaceae) and Carex gayana (Cyperaceae), in 21 high Andean wetlands of Chile’s Norte Chico. Using rbcL gene sequences and AFLP markers, we found that both species displayed low levels of within-wetland genetic diversity, high inter-population genetic differentiation, and spatially-dependent genetic variation arising from isolation-by-distance. The distance at which populations become genetically independent was of the same order of magnitude in both species (125-175 km). Despite these similarities, idiosyncratic spatial patterns were detected. C. gayana in the three most northeastern wetlands demonstrated marked differences relative to the rest of the populations, with the latter group following a latitudinal stepping-stone pattern. In P. clandestina, a genetic barrier was found to divide the northern and southern populations into two balanced groups, and spatial genetic variation was consistent with a hierarchical island model. The data indicate that each of the two species likely responded to different geological and ecological events, resulting in the definition of unique evolutionarily significant units in both. These results suggest that the implementation of global conservation programs at regional scales would likely result in the loss of important components of biodiversity in these ecosystems, and underscore the need for caution in designing effective conservation strategies.