AbstractObjectiveProtecting biodiversity is important for preserving ecosystem functions and services, and understanding the diversity present in a system is necessary for effective conservation. Lampreys display extensive diversity in morphology and life history strategy. The extent of this diversity and the underlying genetic patterns have not yet been fully characterized. Uncertainty about species boundaries and operational taxonomic units hinders lamprey management and conservation. Limited data have been collected on California lampreys (Lampetra spp. and Entosphenus spp.), but evidence suggests widespread population decline across the west coast of North America. Study objectives were to identify which nominal species are present at study sites across northern and central California, determine whether current taxonomic classifications accurately and sufficiently describe lamprey diversity in California, and characterize the biogeographic distribution of genetically distinct lamprey lineages across the study area.MethodsTo achieve these objectives, this study utilized DNA barcoding, phylogenetic analysis, and species delimitation analysis. Lamprey individuals (N = 87) from 19 sites in the Sacramento–San Joaquin River basin, San Francisco Bay, and Klamath River basin were sequenced for the mitochondrial cytochrome b (cyt b) gene, and the data were combined with publicly available lamprey cyt b sequences for analysis.ResultResults showed relatively deep phylogenetic divergence between Lampetra and Entosphenus. Distinct and genetically divergent lineages were observed within Lampetra, while distinct but genetically similar lineages were observed within Entosphenus. This study revealed novel Lampetra lineages in the Napa River and Alameda Creek, and results suggest that the biogeographic distribution of lamprey diversity may follow patterns observed in other native fishes. Species delimitation results indicated that at least seven separate candidate species of Lampetra can be found in California, suggesting that California holds more species‐level diversity than the expected three nominal Lampetra species known to occur in the state.ConclusionThese results highlight the underestimated diversity of lampreys in California and the need for further assessment of taxonomic classifications and operational taxonomic unit designations of California lampreys.