Forensic identification techniques used in cases involving animals, animal abuse or co-mingled at crime scenes often rely on blood or shed hair analysis to identify the species. While there are qPCR or STR commercial kits available for DNA testing of domestic animals, some only test for one animal species at a time. The qPCR kits are used in food fraud cases to test if a 100% beef hot dog may actually contain other meats and can identify that adulteration (Ali, et al, 2014, Ali et al, 2015). However, if the food was adulterated with lamb, for example, a species-specific kit for chicken would not be able to confirm that mixture of meat products. In other examples, crime scenes often find hair, tissue, or blood at the scene. Common presumptive tests will test for human evidence. But the scene can often be mixed with non-human DNA evidence—evidence that could provide additional leads in a case. However, to date, no presumptive method provides a rapid identification of multiple unknown species as the source of the evidence—critical to know before moving to STR analysis. This project used mitochondrial DNA cytochrome B gene marker, to create a reference ladder and design two multiplex kits that can simultaneously detect dog, cat, rabbit, donkey, goat, sheep, chicken, pig, cow, horse and human DNA using a traditional forensic workflow. Labeled universal forward primers and species-specific reverse primers were used for simultaneous amplification of 11 species in two multiplexes (5, 6). The individual species' PCR products were column-cleaned and then mixed to form a reference ladder with bin sets for precise identification of a sample. The validation of the ladder and kits were tested against mock crime scene samples of blood found in soil, on wood, carpeting and clothing. In addition, two undergraduate interns tested the kits on various dog food samples to verify ingredients on the labels did indeed match the meat products used in the production of the kibble. Ongoing research continues to expand the reference ladder by adding rat, mouse, guinea pig and ferret species. Additional reference ladders for wildlife, fish, and plants will be developed using the same protocols in the future