Predation by the red fox Vulpes vulpes is believed to be threatening the little penguin Eudyptula minor on Phillip Island in Victoria. Polymorphism at seven microsatellite loci was examined to estimate the extent of differentiation between Phillip Island and mainland populations of V. vulpes. Loss of alleles has occurred on Phillip Island where foxes first appeared approximately 88 years ago compared with mainland populations. Genetic differentiation between the Phillip Island and mainland populations was high. The relatively high differentiation found between the two populations could be due to either low migration rates, the effect of the composition of founder animals or both effects. Further ecological and historical information about the populations is needed to explore the likely significance of these effects.