Sequence divergence for a fragment of the 16S rRNA gene was compared to identify the advantages in using mitochondrial genes that descend separately through the female and male lineages to examine population structure. The test compared divergence among four local species of freshwater mussels (Unionidae) and was extended to multiple populations of one species, Pyganodon grandis. For the same gene, the male-inherited sequences diverged at a faster rate, producing longer branch lengths in the phylogenies. Of particular use were sequences extracted from P. grandis populations from the southern region of the Lake Erie watershed (Ohio, USA); five male-inherited haplotypes were found. Only one change was observed in the female-inherited form in this region. Therefore, more rapid evolution has occurred in the male form of the gene, and this form provided stronger evidence of geographical isolation among populations. A combination of analyses on haplotypes derived through males and females creates complementary opportunities to identify evolutionary relationships caused by drift and migration in mussels.