The family Margaritiferidae is a small but widely distributed group within the Unionoida, or freshwater mussels, whose taxonomy and systematics has been the subject of numerous publications. Despite several efforts, there is no consensus on which characters reliably diagnose this family. Herein, we present the results of a phylogenetic analysis of the most comprehensive data set for Margaritiferidae in terms of taxa and phylogenetic markers assembled to date, including eleven out of the twelve margaritiferid species currently considered valid. In addition, we review the fossil record of the family and attempt to integrate fossil and DNA sequence data to provide a diagnosis of Margaritiferidae, identify its origin and biogeographic patterns, and determine the systematic relationships of its constituent species and their taxonomic affinities. We assembled a molecular data set comprised of five markers: COI, 16S, 28S, 18S and histone 3 for a total of 59 specimens representing eleven species of Margaritifera. Our results indicate that the family Margaritiferidae is a monophyletic group comprised of the single genus Margaritifera, which includes the following 12 species: M. dahurica, M. margaritifera, M. monodonta, M. middendorffi, M. laevis, M. marrianae, M. hembeli, M. falcata, M. laosensis, M. auricularia and M. marocana plus the unstudied M. homsensis. Estimates of divergence times using fossil calibrations or mean substitution rates produced dramatically different results. Divergence estimates based on the fossil calibrations were 10 times higher than those obtained applying the mean substitution rates. The current distribution of the family implies dispersal across marine or brackish waters by their host fish, leaving a fossil record on four continents that dates to the Mesozoic. Margaritiferidae appear to be derived from putative ancestor in the Silesunionidae, with a likely origin in Asia. We suggest that Margaritiferidae had spread along the Tethys margins and crossed the Atlantic already in the Late Triassic or Early Jurassic. Further dispersal events, in the Late Cretaceous or Eocene, may be linked to salinity‐depleted coastal waters or freshwater layering.