In Little Penguins Eudyptula minor there are no reliable plumage or body size differences that can be used visually to distinguish the sex of individuals. However, sexual dimorphism of morphometric measures has been noted, with males always being a little larger than females. In this study, differences between E. minor sexes at eight colonies in south-eastern Australia were determined statistically via discriminant function analysis (DFA) and through the utilization of DNA-based techniques developed for non-ratite birds. The DFA correctly determined gender in 91.1% of cases and molecular methods were 100% accurate. Our DFA success rate of classification is similar to that previously published for Little Penguins in Victoria. In this study statistically significant differences in mean bill depths and lengths were found between Little Penguin colonies at St Kilda, Phillip Island and Gabo Island, compared to colonies at Kangaroo Island, Granite Island, Middle Island and London Bridge. As birds in eastern populations (St Kilda, Phillip Island, Gabo Island) exhibit statistically significantly smaller beaks (bill depth and bill length), separate discriminant functions were investigated for each phenotypically distinct geo-spatial cohort. Interestingly, cluster analysis for bill length identified three groups: western (Kangaroo Island and Granite Island), eastern (St Kilda, Phillip Island and Middle Island) and the London Bridge Little Penguin colony, which constituted a separate group. We conclude that while there is a slight increase in DF power for colonies west of Cape Otway and for some specific colonies, colony-specific DFA is not required to identify the sex of Little Penguins in south-eastern Australia.