- Fish monitoring programs often rely on the collection, species identification, and counting of individual fish over time to inform natural resource management decisions. Thus, the utility of the data used to inform these decisions can be negatively affected by species misidentification. Fish species misidentification bias can be minimized by confirming identification using genetic techniques, training observers, or adjusting monitoring data using estimates of incomplete detection and false-positive misidentification. Despite the existence of well-established fish identification training and quality control programs, there is considerable uncertainty about fish species false-positive misidentification rates and the effectiveness of fish identification training programs within the San Francisco Estuary. We evaluated the misidentification of fish species among Delta Juvenile Fish Monitoring Program observers by conducting five fish identification exams under controlled conditions at the Lodi Fish and Wildlife Office in Lodi, California, between 2012 and 2014. To assess the variability in false-positive misidentification, we fitted data to species and observer characteristics using hierarchical logistic regression. We found that fish species misidentification was fairly common, averaging 17% among 155 test specimens and 32 observers. False-positive misidentification varied considerably among species and was negatively related to fish size, the abundance of the species within monitoring samples, and observer experience. In addition, observers who were not formally trained or used as full-time observers were, on average, 6.0 times more likely to falsely identify a species. However, false-positive misidentification rates among observers and specimens still varied considerably after controlling for observer experience and training, and species and size, respectively. Our results could be used to improve fish identification training and testing, increase the accuracy of fish occupancy or abundance estimation, and justify the allocation of resources to continually use and formally train full-time observers within long-term monitoring programs operating in the system.