Polymerase chain reaction (PCR) diagnostic tests are increasingly applied to the identification of wildlife. Yet rigorous verification is rare and the estimation of test accuracy (the probability that true positive and true negative samples are correctly identified - test sensitivity and specificity, respectively), particularly in combination with sequencing, is uncommon. This is important because PCR-based tests are prone to contamination in sampling and the laboratory. Here, we use an experimental case-control approach to estimate the sensitivity and specificity of a sequential PCR-based wildlife detection test used to identify incursions of red foxes into Tasmania from predator faeces (scats). Our results show that the sensitivity of the fox test is high (~94%) for the PCR-based test on its own, but this decreases to ~84% when combined with the DNA sequencing step. In contrast, the specificity increases from ~96% in the PCR-only test to 99·6% after inclusion of the DNA sequencing step. The intense public scrutiny of the fox eradication programme in Tasmania has undoubtedly influenced the application of a sequential PCR test that maximizes specificity at the expense of sensitivity and so increases the risk that scats containing fox DNA would not be detected. This could lead to the establishment of foxes in Tasmania as a consequence. Synthesis and applications. Importantly, the estimation of the sensitivity and specificity of sequential tests enables decisions about the risk associated with mistaken identification (i.e. false negatives vs. false positives) to be quantified for decision-makers. The cost of false-negative errors should be balanced against the costs of false-positive errors, which could include the expenditure incurred in the application of unnecessary management actions were foxes not in fact present. Understanding the risks and costs associated with both false-negative and false-positive errors is therefore a key component to the decision-making process for the management of the Tasmanian fox incursion.