The geographically closed, non-migratory populations of endangered Laysan Teal (Anas laysanensis) were sampled in the wild. This species was once widespread across the Hawaiian archipelago, but became isolated on Laysan Island (415 ha) from the mid-1800s until 2004 when a translocation to Midway Atoll (596 ha) was undertaken to reduce extinction risks. We compared genetic diversity and quantified variation at microsatellite loci sampled from 229 individuals from the wild populations at Laysan Island (1999–2009) and Midway Atoll (2007–2010; n = 133 Laysan, n = 96 Midway birds). We identified polymorphic markers by screening nuclear microsatellites (N = 83). Low nuclear variation was detected, consistent with the species' insular isolation and historical bottleneck. Six of 83 microsatellites were polymorphic.