An understanding of the calling behaviour of nocturnal birds is critically important for their detection. The Australian Masked Owl (Tyto novaehollandiae) is most commonly located by call playback surveys that prompt calling responses by resident owls, yet Masked Owl calling behaviour remains poorly understood. We present a detailed analysis of the calling behaviour of the Tasmanian subspecies of the Australian Masked Owl (T. n. castanops) to, (1) describe the vocal repertoire of the species, using spectrograms, (2) examine differences in relation to plumage colour morph, sex, age and individual variation to consider the potential for identifying individual birds, and, (3) examine environmental and temporal effects on calling behaviour. Three types of calls were identified: screech, continuous screech and chatter calls. The Tasmanian Masked Owl was found to produce a deeper (lower frequency) screech than mainland Masked Owls. Adult and juvenile Masked Owls produced different screech calls, and paired male and female Masked Owls produced different chatter calls from each other. Comparison of the chatter calls between individuals suggested that these types of calls may be used to identify individual owls via this non-invasive call playback survey technique.