Jabiru Stork (Jabiru mycteria is listed under Appendix I of CITES and considered threatened in Central America. The first population genetic analysis of Jabiru Storks was carried out using mitochondrial DNA (mtDNA) control region sequences (520 bp) and five heterologous microsatellite loci. Samples were collected from the field (N = 49) and museum skins (N = 22) in Central (mainly Belize, Nicaragua and Costa Rica) and South America (Colombia, Venezuela, Peru and Brazil). A decline of mtDNA diversity was observed in comparisons between past (N = 20) and present (N = 40) samples collected in Central America and northern South America. Similar levels of microsatellite loci diversity were observed among contemporary samples. Lower levels of mtDNA variability were observed in samples from Central America and northern South America when compared to the Brazilian Pantanal region. Significant levels of genetic differentiation were found between contemporary locations sampled, whereas non-significant results were observed for historic samples. The non-geographic association of haplotypes observed at the cladograms and the recent divergence times estimated between locations are indicative of an evolutionary history of a large population size with limited population structure. Reconnection of populations via increased gene flow, particularly in Central America, is recommended if genetic structure and status are to be restored.