Unio tumidus is a native European freshwater mussel from the family Unionidae. These mussels have a unique system of mitochondrial DNA inheritance called doubly uniparental inheritance (DUI). Under DUI, two types of mitochondrial DNA are present: haplotype F (female genome)—inherited from mother and haplotype M (male genome)—inherited from fathers to male offspring. The F genome occurs in eggs and in somatic tissues of both sexes, whereas the M genome is present in male gonads and gametes. We characterized three M and three F mitochondrial genomes of Unio tumidus. The lengths of these genomes were 15769–15770 bp for F type and 16607 bp for M type. In both genomes, the set of 38 genes which is typical for Unionidae was identified: 37 metazoan genes and gender-specific ORFs. The non-coding sequences constituted only 5.2 and 3.5% of F and M genome, respectively. Both genomes were similarly high in average AT content (65–66%) but intraspecific nucleotide diversity amongst the three M genomes of U. tumidus was four times lower than amongst F genomes. The patterns of polymorphisms across the mitogenomes of the closest relatives confirmed that the M genomes accumulate more substitutions and the conserved regions within one lineage are also conserved in the other.