Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Parides ascanius (Lepidoptera: Papilionidae) is a butterfly endemic to the sand forests ("restingas") of one of the most populated areas of Brazil (from Rio de Janeiro state to South Espirito Santo state), and was the first invertebrate officially recognized as being threatened in Brazil. Here we present a panel of eight polymorphic microsatellite loci and partial sequences of mitochondrial gene COI aiming to characterize this butterfly's genetic diversity and understand its distribution among the extant populations. We estimate F-ST metrics, migration rates, cluster assignment, and spatial structure of genetic diversity. F-ST and statistics indicate low genetic structure and no evidence for endogamy, with all populations connected by high migration rates. Seven populations have low permanence rates (68-75 %) with increased migration probabilities for all populations. One population displays higher permanence rate (87.7 %), as the metropolitan matrix isolates it. Spatial analysis shows a global structure around the city of Rio de Janeiro and the Guanabara Bay; assignment analysis recovers six clusters evenly spread among sampled populations. These findings are consistent with a natural scenario of metapopulation dynamics for P. ascanius, with low genetic diversity and no endogamy, but progressively isolated by the metropolitan matrix. Conservation efforts should focus in connecting the isolated population, broaden the searches for new populations, and preserve all extant habitat patches where P. ascanius still occurs.173533546Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Brazil [141254/2013-0]Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior, Brazil [3700/14-3]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Brazil [2008/52197-4]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Brazil Grant from BIOTA/FAPESP [2011/50225-3]Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)