To date, no PCR diagnostic exists to directly identify mammalian blood meals from mosquitoes by sized DNA fragments following agarose gel electrophoresis. We have developed a vertebrate-specific multiplexed primer set based on mitochondrial cytochrome b to identify the mammalian blood hosts of field-collected mosquitoes. Although designed for the study of African malaria vectors, the application of this tool is not restricted to this disease system. Validation of this diagnostic on dried anopheline and culicine field specimens collected in Zambia and Mali demonstrated that blood meals could be identified two to seven months after collection. Time course experiments revealed that host DNA was detectable in frozen mosquito abdomens 24-30 hours post-feeding. Additionally, multiple blood meals from different mammals could be detected in a single mosquito. This diagnostic will be a valuable tool for identifying the blood meals of field-collected mosquitoes where people and alternative mammal hosts are present.