We isolated and characterized eight novel microsatellite loci in the little penguin Eudyptula minor, using nonradioactive polymerase chain reaction-based techniques to screen GA and GAAA repeats from enriched genomic DNA libraries. All eight loci were polymorphic and seven were variable in our main study population. Cross-amplification using a microsatellite primer developed in Spheniscus demersus(African penguin) yielded one additional polymorphic locus. This locus combined with six of the little penguin loci is suitable for paternity assignment in little penguins.