In marine pollution monitoring, the biomarkers recorded in sentinel organisms are influenced by natural confounding factors that may jeopardise their interpretation. Among these confounding factors, little is known about the influence of sex along the annual reproductive cycle. The present investigation aims at contributing to understand how sex and sex-related differences in gamete development progression impinge on biomarker baseline values and on biomarker responsiveness to pollution in sentinel mussels. Mussels (Mytilus galloprovincialis) were collected from a relatively clean locality and from a chronically polluted site in the Basque Coast (Bay of Biscay) in January, April, August and November. Sex and gametogenesis stages were determined for each mussel. Tissue concentration of metals and PAHs was analysed. A battery of biomarkers was investigated: cytochrome c oxidase, pyruvate kinase and phosphoenolpyruvate carboxykinase enzyme activities; levels of protein carbonyls, malondialdehyde and 4-hydroxy-2-nonenal; lysosomal enlargement and membrane stability; intracellular neutral lipid accumulation; cell type composition and thinning of the digestive gland epithelium; and survival-in-air. Sex- and reproductive stage-related differences were found in bioaccumulation and in the values and responsiveness of most of the biomarkers. However, the patterns of sex-related differences were not consistent across all biomarkers. The differences in the biomarker responses between females and males also depended on the season, reflecting the progression of the gametogenesis cycle. Thus, selecting mussels of one specific sex does not seem to be a crucial requisite to carry out biomarker-based monitoring; yet, it is highly recommended to identify sex condition and gamete developmental stage of each mussel to test for the potentially confounding effects of sex, reproductive status and sex-related variability along the reproductive cycle.