Magellanic Penguins (Spheniscus magellanicus) show little sexual dimorphism, and although males are usually larger than females, sexing by direct observation may be difficult, especially in the case of chicks. In this paper we evaluate the utility of four different PCR-based sex determination techniques using genomic DNA for sexing Magellanic Penguins. We found that the primer set designed for sex determination in Collared Flycatcher (Ficedula albicollis) also provided a reliable, simple and convenient sexing procedure for Magellanic Penguins. Additionally, we obtained discriminant functions for sexing adults and chicks, sampled at six colonies differing in size and other ecological characteristics. Discriminant function for adults used two variables, bill length and bill depth that correctly classified 97% of the birds. Discriminant function for chicks included bill length and flipper length and correctly classified 78% of the individuals. Although molecular and biometric approaches could be useful for sexing adult Magellanic Penguins, only molecular procedures proved appropriate for accurately sexing chicks.