An identification assay has been developed that allows accurate detection of 19 of the most common terrestrial mammals present in New Zealand (cow, red deer, goat, dog, horse, hedgehog, cat, tammar wallaby, mouse, weasel, ferret, stoat, sheep, rabbit, Pacific rat, Norway rat, ship rat, pig, and brushtail possum). This technique utilizes species-specific primers that, combined in a multiplex PCR, target small fragments of the mitochondrial cytochrome b gene. Each species, except hedgehog, produces two distinctive species-specific fragments, making the assay self-confirmatory and enabling the identification of multiple species simultaneously in DNA mixtures. The multiplex assay detects as little as 100 copies of mitochondrial DNA, which makes it a very reliable tool for degraded and trace samples. Reliability, accuracy, reproducibility, and sensitivity tests to validate the technique were performed. The technique featured here enabled a prompt response in a predation specific event, but can also be useful for wildlife management and conservation, pest incursions detection, forensic, and industrial purposes in a very simple and cost-effective manner.