The vocal characteristics of a species can be immensely diverse, and can significantly impact animal social interactions. The social structure of a species may vary with geographical variation in call characteristics. The ability of pinnipeds (true seals, fur seals, sea lions and walrus) to distinguish between conspecifics may assist male reproductive strategies, particularly mate acquisition. We assessed the ability of mate guarding Australian sea lion (Neophoca cinerea) males to discriminate local from foreign males’ barks recorded from a geographically distant breeding colony. Bark characteristics were significantly different between colonies, with barks produced by males from the Lewis Island breeding colony higher pitched and longer in both duration and interval duration than barks produced by males on Kangaroo Island. Mate guarding males displayed inter-colony discrimination of barks, with a significantly stronger response to barks from local males than to those of males from a colony approx. 180 km away. Local males’ barks were apparently considered a greater threat than barks from unfamiliar males. We propose that discrimination of acoustic characteristics may facilitate reproductive isolation in this species that may lead to an ethological–acoustic barrier between breeding colonies, and subsequent genetic isolation.