Doubly uniparental inheritance (DUI) of mitochondrial DNA (mtDNA) implies the coexistence of two highly divergent gender-associated mitogenomes, which are transmitted independently. This exception to the strictly maternal inheritance of mtDNA is characteristic of some bivalves and occurs in the endangered freshwater mussel Margaritifera margaritifera (Linnaeus, 1758). This study identifies for the first time new male 16S rDNA and cox2-cox1 haplotypes of M. margaritifera by using non-destructive sampling of haemolymph. Samples collected from mussels with spent gonad contained exclusively the female (F) mitogenome, whereas haemolymph extracted from reproductively active mussels can be used to procure the male (M) mitogenome. Identification of partial 16S rRNA secondary structures of female and male sequences in M. margaritifera supported the existence of structural differences between the two gender-associated transcripts. Moreover, the novel 16S rDNA and cox2cox1 sequences were integrated into a phylogenetic tree including homologous mtDNA sequences from several unionoid species. The phylogram revealed two F and M clades and haplotypes clustered according to the family taxa, with species of the family Margaritiferidae occupying a basal position. The findings are consistent with the hypothesis of a single evolutionary origin for the DUI system in bivalve freshwater mussels. Both 16S rDNA and cox2-cox1 markers may be useful for investigating gender-associated diversity across the geographical range of this endangered species with repercussions for genetic sexing of wild and captive mussels. (C) 2016 The Linnean Society of London, Zoological Journal of the Linnean Society, 2017