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Abstract: In recent times, there has been an increased concern over the appearance of human estrogens in marine ecosystem and their effects on the marine habitat. Discharge of raw sewage has been identified as one of the most important sources of human estrogens in the marine environment. Therefore, we have developed a gas chromatography–(ion-trap) mass spectrometry/mass spectrometry method for the analysis of natural estrogens estrone (E1), and 17β-estradiol (E2) and synthetic estrogens 17α-ethynylestradiol (EE2) and diethylstilbestrol (DES) in sewage effluents, seawater and mussels. Recovery of target analytes from mussels (n =3) was above 60% with RSD ranging from 8% to 13%. For aqueous samples (n =3) recoveries were above 80% with RSD ranging from 3% to 7%. Method detection limits for the target analytes ranged from 0.1ngg−1 to 1.0ng/g for mussel sample analysis and from 0.5ngL−1 to 1.2ngL−1 for water sample analysis. The usefulness of the method was demonstrated by analyzing environmental samples from St. John’s and Halifax, Canada, where raw sewage is directly discharged into the harbors. Estrone and 17 β-estradiol were found at 1.5ngL−1 and 1.8ngL−1 in seawater samples collected from St. John’s harbor, while trace amounts of estrone was measured in some mussels collected from Halifax harbor. [Copyright &y& Elsevier]