The orchid genus Nigritella constitutes a polyploid complex which is widespread in mountain regions in Europe. Diploid members of the genus have sexual reproduction, whereas polyploid members are characterised by agamospermy. We used allozyme data to estimate levels of variation at different hierarchical levels and to describe the evolution of polyploids. - The variation patterns at allozyme loci agree with the mode of reproduction. Thus, populations of diploid species are variable, whereas populations of polyploid species contain one or two multilocus genotypes. The two tetraploids N. widderi and N. miniata contained two different multilocus genotypes each, indicating either multiple origins, or else sexual recombination or mutation at the tetraploid level. - The two tetraploids N. nigra subsp. austriaca and N. nigra subsp. iberica are closely related to the triploid N. nigra subsp. nigra, and they may have evolved by hybridization of this triploid and a diploid species. - In agreement with previous data, allozyme data confirm that the tetraploid apomict Gymnigritella runei is formed by fusion of an unreduced gamete from N. nigra subsp. nigra with a normal, haploid gamete from Gymnadenia conopsea. - The multilocus genotype found in Nigritella archiducis-joannis was identical to one multilocus genotype found in N. widderi, indicating that they may have evolved from a similar set of parental taxa. The pentaploid N. buschmanniae may be derived by hybridization of N. widderi with a sexual diploid species. - The multilocus genotype found in N. widderi was identical to one of the multilocus genotypes found in N. miniata, indicating a close relationship of these taxa as well. - The polyploid species investigated appear to combine divergent genomes and are likely to be derived by allopolyploidization. They all contain alleles that are rare or absent from present-day diploids, indicating that the polyploid taxa are derived from extinct ancestors and that they may have evolved at least before the last glaciation. - A comparison with two species of Gymnadenia, G. conopsea and G. odoratissima, revealed that Gymnadenia and Nigritella are more divergent from each other than species within each genus, which agrees with the view that the genera are sister groups.