Using 32 allozyme loci, we examined the genetic diversity of ten populations of Neottia ovata differing in size and located in different regions of northeast Poland. Within‐population genetic variation was low (Ppl = 9.4–31.2%, A = 1.09–1.34 and HE = 0.044–0.128) relative to taxa with similar life histories. In the majority of N. ovata populations (excluding those in Biebrza), we noted high genotypic diversity (0.49−1.00) and high proportions of unique genotypes (86–100%). Neottia ovata shows a strong pattern of genetic differentiation among populations in northeast Poland, reflected in the high overall FST value (0.409). There was a weak, but significant, relationship between genetic and geographical distance (r = 0.09, P < 0.05). We discuss the breeding system, small population sizes and population subdivision as the most important factors affecting the genetic diversity of this species. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170, 40–49