| Summary: | Abstract When analyzing environmental samples for DNA from multiple taxa, researchers must usually decide between iterative analyses with single‐taxon assays—which are reliable and sensitive, but also laborious to apply—and approaches such as metabarcoding that can simultaneously target multiple species, but which are less sensitive for detection across taxa. Here, we test an intermediate approach that allows efficient, parallel assessment of taxon‐specific qPCR assays via high‐throughput quantitative PCR (HT‐qPCR). Based on an assessment of over 500 environmental samples, we found that sensitivity and specificity of our HT‐qPCR approach were similar (concordance 0.900–1.000) to values achieved through single‐species qPCR in six out of seven assays tested. Thus, HT‐qPCR may provide analyses of similar quality as single‐species qPCR analyses for environmental DNA, but at a lower cost per taxon. We see this approach as being a valuable addition to the eDNA sampling toolbox, particularly for situations where reliable inferences are needed for a defined suite of rare invasive or imperiled taxa.
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