Malaxis monophyllos is a rare orchid with a fragmented boreal-montane distribution in Europe where it is associated with both natural swampy and anthropogenic habitats. We employed extensive sampling of M. monophyllos populations from different habitat types, over its whole European geographic range, to examine its genetic diversity patterns and phylogeographic structure using amplified fragment length polymorphisms (AFLPs). Our results revealed the relatively low genetic diversity of M. monophyllos, with the effect of small population sizes and inbreeding as the driving forces operating within the European part of its range. The statistically highest values of genetic diversity were found in populations from the boreal region (average: percentage of polymorphic loci PPL₃ = 21.6%, Nei's gene diversity H_j3 = 0.144, the rarity index DW₃ = 1.34), while populations from mountainous regions were characterised by a reduced level of genetic diversity (e.g. an average for Alpine populations: PPL₃ = 18.1%, H_j3 = 0.121, DW₃ = 0.84) in comparison to boreal ones. Our results revealed that the newly established anthropogenic populations in the Polish uplands were probably founded from numerous external sources and should be considered a significant source of the species' genetic diversity. We also confirmed the low genetic differentiation among M. monophyllos populations (F_ST = 0.074), with the lack of distinguishable genetic clusters, that supports results about the multidirectional gene flow between M. monophyllos populations in Europe, and directed conservation efforts on conserving all suitable for this species habitats.