Many endangered plant species suffer from the effects of ongoing fragmentation of their populations leading to a loss of genetic diversity through genetic drift. In addition, populations of rare plants may also be affected by hybridization with other plant species. We studied the genetic population structure of populations of the endangered Gladiolus palustris in the western limit of its distribution area using AFLP markers. In addition, we clarified the taxonomic status of populations where hybridization with the closely related G. imbricatus was suspected based on morphology by sequencing the ITS region of the nuclear ribosomal DNA as well as two regions of the chloroplast DNA. Our analysis of the genetic population structure of G. palustris revealed a strong differentiation among geographical regions, which was much higher than the differentiation among populations within regions. Most populations retained a considerable amount of genetic variation. To counteract the future loss of genetic diversity through drift, we recommend using the largest populations per region as seed source to increase genetic diversity in genetic depauperate populations of the same region. Our genetic analyses indicated that some French populations of G. palustris are of hybrid origin. These hybridization events are likely to be ancient as these populations are very isolated and some parent taxa went regionally extinct. As these hybrid populations may elucidate post-glacial distribution patterns of related Gladiolus taxa, we advocate that they deserve the same conservation efforts as purebred ones.