We examined sexual dimorphism in bill size in adult Adelie Penguins Pygoscelis adeliae, adult Chinstrap Penguins P. antarctica and adult and juvenile Gentoo Penguins P. papua at King George Island, Antarctica, using a DNA-based molecular sexing technique. Bill length and depth were the most consistent dimorphic character examined, with measurements 5.4%–11.5% larger in males than in females, on average. Within breeding pairs sampled, male Chinstrap and Gentoo Penguins had consistently longer and deeper bills than their mates, although bill measurement overlapped between sexes at the population level. We used bill measurements to calculate species- and age-specific discriminant functions that correctly classified 83.2%–96.7% of the individuals in our study following cross-validation. The discriminant functions derived from this analysis provide a practical method of sex determination for all three Pygoscelis penguin species in the South Shetland Islands where they breed sympatrically. Posterior probability analysis can also be used to identify individuals that are likely to be incorrectly classified using discriminant function analysis, allowing DNA-based tests for gender to be reserved for targeted use. Furthermore, we report raw morphometric data to facilitate future analysis and discriminant function improvement.