Cross-species amplification using domestic cat microsatellite primers is the primary method used for nuclear genetic research in lions (Panthera leo). Genetic differences introduced over 10.8 million years of divergence between the two species make these markers problematic when using low quality and low quantity DNA, a common issue in wildlife genetic research. To increase amplification success of microsatellites in the lion, miniSTRs (<150 bp) with primers designed to be closer to the target region and specific to the lion were developed. Lion specific STRs were successfully designed for 14 of 17 commonly used microsatellites, 10 of 14 being miniSTRs, all with 100% amplification success when tested on 30 lion samples with DNA of varying quality and quantity.