AbstractRiver herring (Alewives Alosa pseudoharengus and Blueback Herring A. aestivalis [bluebacks]) are congeneric, anadromous clupeid fishes that hybridize in areas of sympatry. Peritoneal pigmentation is the most diagnostic characteristic used to distinguish the species, but pigmentation has not been examined in hybrids. We developed a molecular‐based assay to distinguish purebred river herring species from F1 hybrids and compared the peritoneal pigmentation among captive‐raised purebred and hybrid individuals. Both wild‐caught Alewife and blueback females tank‐spawned with conspecific and congeneric males, but Alewives required exogenous hormone administration. Larvae of both species and hybrids were raised for ∼290 d posthatch, and peritoneal pigmentation was quantified. A nuclear gene (rag2) restriction fragment length polymorphism assay was used for species and hybrid identification. The peritoneal pigmentation was significantly darker in hatchery‐spawned bluebacks than in Alewives, and hybrids exhibited consistent, intermediate expression. Adult individuals collected from the wild, however, exhibited darker (Alewives) or more variable (bluebacks) peritoneal coloration than their respective, captive‐reared juveniles. These results indicated that peritoneal pigmentation alone is insufficient for diagnostic river herring species identification and should be coupled with genetic assays.