In keeping with many species of waterbirds, there is little difference in the body size or external appearance of adult male and female Northern Gannets (Morus bassanus). Previous work has indicated that some morphological characteristics and behavioral patterns differ on average between sexes but there has yet to be a quantitative evaluation of the accuracy or applicability of these different criteria in determining sex. The aim of this study was to use DNA-based techniques to determine the sexes of adults and to use this information to evaluate a range of behavioral and morphological criteria for sex determination. Nape-biting behavior was frequently observed, was performed almost exclusively (>99% of observations) by males and was completely accurate in assigning sexes on the basis of multiple observations at each nest: this confirms previous results which indicated that nape-biting is highly sex-specific. Collection of nest material was also performed primarily by males, but this seldom occurred and so despite over 500 h of observations at each nest, fewer than 30% of pairs were sexed correctly using this criterion. Only two-thirds of birds were sexed correctly from head plumage color and fewer than 30% from foot web-line color. Behavioral observations were as accurate as DNA-based sexing and more effective than morphology for sexing adult birds at all stages of chick rearing.