The presence of xenobiotics, such as metals, in ecosystems is concerning due to their durability and they pose a threat to the health and life of organisms. Moreover, mercury can biomagnify in many marine food chains and, therefore, organisms at higher trophic levels can be adversely impacted. Although feathers have been used extensively as a bio-monitoring tool, only a few studies have addressed the effect of both age and sex on metal accumulation. In this study, the concentrations of trace elements were determined in the feathers of all members of a captive colony of African Penguins (Spheniscus demersus) housed in a zoological facility in Italy. Tests were performed by inductively coupled plasma-mass spectrometry to detect aluminum, arsenic, cadmium, cobalt, chromium, copper, iron, manganese, nickel, lead, selenium, tin, vanadium, and zinc. Mercury was detected by a direct mercury analyzer. Sexing was performed by a molecular approach based on analyzing the chromo-helicase-DNA-binding1 gene, located on the sex chromosomes. Sex- and age-related differences were studied in order to investigate the different patterns of metal bioaccumulation between male and female individuals and between adults and juveniles. Juvenile females had significantly higher arsenic levels than males, while selenium levels increased significantly with age in both sexes. Penguins kept in controlled environments-given that diet and habitat are under strict control-represent a unique opportunity to determine if and how metal bioaccumulation is related to sex and age.