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• Nationwide PRF seizures reveal 9,479 animals and 9.3 t of wild meat (2017–2024). • Birds dominate in Caatinga and Pampa; mammals supply most biomass overall. • Seizures reflect defaunation: more individuals but lower biomass in degraded states. • Mammal decline linked to substitution by birds; reptiles show distinct transport bias. • Roadway seizure data offers a cost-effective barometer of illegal wild meat use. Illegal and unsustainable wild meat use is simplifying vertebrate assemblages across the Neotropics, yet robust information on offtake magnitude and use patterns remains scarce. We used nationwide road-seizure records from Brazil’s Federal Highway Police (Polícia Rodoviária Federal; PRF) from 2017 to 2024, focusing on hunted animals and wild meat intercepted during transport, to quantify the scale and composition of illegal offtake. We then tested whether these enforcement records reproduce expected ecological signals of illegal hunting by examining whether seizure yield and taxonomic composition track state-level defaunation. Across 314 independent events, PRF intercepted ∼9,479 individual animals, totalling ∼9.3 t of biomass, revealing substantial and taxonomically structured pressure on wildlife. The Amazon and Caatinga biomes accounted for the largest shares of events and individuals, respectively. Seized birds dominated numerically in Caatinga, whereas mammals supplied most biomass overall, with reptiles contributing disproportionately in the Amazon and Cerrado–Pantanal biomes. Generalized linear models showed that with increasing defaunation, events involved more individuals but less biomass per seizure, and the mean body mass of seized vertebrates declined. In addition, the proportion of mammals decreased while birds became more prevalent. This convergence with known depletion patterns indicates that road-seizure data can serve as a cost-effective barometer of illegal wild-meat use and its defaunation-linked “downsizing” effect. Targeted responses should pair highway and riverine enforcement, standardize inter-agency reporting (species, counts, biomass, geolocation, product form), and combine access control with community partnerships, livelihood alternatives, and health-risk communication to reduce demand and long-term ecological and public-health impacts.