The availability of molecular methods for avian sex identification has revolutionised the study of sexual differences in behaviour, morphology, life-history traits and conservation management. We implemented the recommendations of a recent review of DNA-based sex-identification by (1) verifying the sex-specificity and (2) estimating the accuracy of different sex-assignment methods in an apparently monomorphic seabird, the Australasian gannet (Morus serrator). The polymerase chain reaction (PCR) method based on the amplification of the sex-linked chromodomain-helicase-DNA binding gene (CHD) repeatedly assigned the same sex in 96% (n = 27 replicates) and correctly sexed all individuals with known gonadal anatomy (n = 6). PCR and sex-specific restriction fragment length polymorphism (RFLPs) showed agreement for 99.5% of individuals (n = 201). DNA-sexed pairs known to be social mates consisted of a male and a female in 96% of pairs sexed by PCR (n = 77) and 98% of pairs sexed by RFLP (n = 65). DNA-sexed females were in the bottom and males in the top copulatory position in 86% of observed copulations (n = 43 individuals). These results validate assumptions that both membership in social pairs and different copulatory positions can serve as reliable behavioural proxies for field-based sex identification in this colonial and obligately biparental seabird.