Relationships among the species of Northern Hemisphere smelts (family Osmeridae) have long been debated in the fish systematics literature. Eight independent studies based on morphological characters failed to reach any consensus on osmerid interrelationships. We reconstruct the osmerid phylogeny based on DNA sequence data from three mitochondrial (cytb, 16S, 12S) and three nuclear (ITS2, S71, RAG1) gene regions from multiple individuals of the 14 species in 6 genera, using the Japanese ayu (Plecoglossus altivelis) as the outgroup. Analyses with different combinations of nuclear and mitochondrial datasets yielded a generally well-resolved phylogeny of the genera that conflicts with previous hypotheses of osmerid interrelationships, and Shimodaira-Hasegawa tests suggest our topology with the current molecular dataset is significantly better than earlier reconstructions. In addition, mapping 114 morphological characters used in previous studies onto our phylogeny shows widespread homoplasy, which is likely the source of the systematic disagreement produced in earlier works.