Cataloging biodiversity is of great importance given that habitat destruction has dramatically increased extinction rates. While the presence of cryptic species poses challenges for biodiversity assessment, molecular analysis has proven useful in uncovering this hidden diversity. Using nuclear microsatellite markers and mitochondrial DNA we investigated the genetic structure of Klamath speckled dace (Rhinichthys osculus klamathensis), a subspecies endemic to the Klamath–Trinity basin. Analysis of 25 sample sites
within the basin uncovered cryptic diversity including three distinct genetic groups: (1) a group that is widely distributed throughout the Klamath River mainstem and its tributaries, (2) a group distributed in the Trinity River, the largest tributary to the Klamath River, and (3) a group identified above a 10 m waterfall in Jenny Creek, a small tributary to the Klamath River. All groups were resolved as divergent in nuclear microsatellite analysis and exhibited levels of divergence in mitochondrial DNA that were comparable to those observed among recognized Rhinichthys species. No physical barriers currently separate the Klamath and Trinity groups and the precise mechanism that generated and maintains the groups as distinct despite contact and hybridizationis unknown. The present study highlights the importance of incorporating molecular analysis into biodiversity research to uncover cryptic diversity. We recommend that future biodiversity inventories recognize three genetically distinct groups of speckled dace in the Klamath–Trinity Basin.