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The moose is a highly prized game species in North America and is often the target of illegal harvesting. Forensic DNA analysis can be used to assist in investigations into wildlife crime by providing the link between the illegal incidents and suspects. In this study, we present the development and validation of a short-tandem repeat (STR) based forensic DNA test for the individualization of moose in Alberta and Yukon, Canada. We show that the markers used in the test are appropriate for forensic use and are sufficiently specific to moose. We also demonstrate the limit of detection and quantitation of the moose STR test on the ABI 3500 genetic analyzer. Lastly, we describe the population genetic structure of moose present in our forensic population database and make recommendations regarding the calculation of appropriately conservative forensic statistics.