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Simple Summary: Understanding the biodiversity of urban ecosystems is critical for management of invasive and pest species, conserving native species, and disease control. Mosquitoes (Culicidae) are ubiquitous and abundant in urban ecosystems, and rely on blood meals taken from vertebrates. We used DNA from freshly blood-fed mosquitoes to characterize the diversity of vertebrate host species in the San Juan Metropolitan Area, Puerto Rico. We collected two mosquito species that fed on a variety of vertebrates. Culex quinquefasciatus fed on 17 avian taxa (81.2% of blood meals), seven mammalian taxa (17.9%), and one reptilian taxon (0.85%). Aedes aegypti blood meals were from a less diverse group, with two avian taxa (11.1%) and three mammalian taxa (88.9%) identified. Domestic chickens dominated the blood meals of Cx. quinquefasciatus, both temporally and spatially, and no statistically significant shift from birds to mammals was detected. The species we detected from the mosquito blood meals provided a snapshot of the vertebrate community in the San Juan Metropolitan Area, most of which were domestic species. However, we also identified a variety of native and nonnative wild species. These results add knowledge about potential ecological factors that impact vector-borne disease management in urban habitats. Urban ecosystems are a patchwork of habitats that host a broad diversity of animal species. Insects comprise a large portion of urban biodiversity which includes many pest species, including those that transmit pathogens. Mosquitoes (Diptera: Culicidae) inhabit urban environments and rely on sympatric vertebrate species to complete their life cycles, and in this process transmit pathogens to animals and humans. Given that mosquitoes feed upon vertebrates, they can also act as efficient samplers that facilitate detection of vertebrate species that utilize urban ecosystems. In this study, we analyzed DNA extracted from mosquito blood meals collected temporally in multiple neighborhoods of the San Juan Metropolitan Area, Puerto Rico to evaluate the presence of vertebrate fauna. DNA was collected from 604 individual mosquitoes that represented two common urban species, Culex quinquefasciatus (n = 586) and Aedes aegypti (n = 18). Culex quinquefasciatus fed on 17 avian taxa (81.2% of blood meals), seven mammalian taxa (17.9%), and one reptilian taxon (0.85%). Domestic chickens dominated these blood meals both temporally and spatially, and no statistically significant shift from birds to mammals was detected. Aedes aegypti blood meals were from a less diverse group, with two avian taxa (11.1%) and three mammalian taxa (88.9%) identified. The blood meals we identified provided a snapshot of the vertebrate community in the San Juan Metropolitan Area and have potential implications for vector-borne pathogen transmission. [ABSTRACT FROM AUTHOR]