Stratiform Zn-Pb deposits hosted in unmetamorphosed carbonaceous and siliceous mudstones of the Ordovician to Silurian Duo Lake Formation define the Howards Pass district in Yukon Territory and Northwest Territories, western Canada. Collectively, the deposits are amongst the largest in the world, containing drill-indicated and inferred resources of 423 Mt at 4.84 % Zn and 1.59 % Pb. Sulphide textures include (a) fine-scale laminations of sphalerite, galena, and pyrite from <0.05 mm to 1 cm thick, interbedded with carbonaceous sedimentary rock; (b) layers of coarse sulphide that are structurally controlled by microfolds; and (c) veins that cut bedding and sulphide laminations. The finely interlaminated nature of sulphides with mudstone has been used as evidence for syngenetic mineralizing processes, whereas paleomagnetic data determined on coarse layered sulphides suggest a Middle Jurassic age of mineralization. Here, we present new rhenium-osmium (Re-Os) isotopic data for 12 pyrite separates obtained from 4 laminated sulphide-rich samples from the XY Central (XYC) and Don (DON) deposits and for 1 unmineralized organic-rich mudstone ∼20 m stratigraphically below the sulphide-bearing zone. Pyrite separates that lack mudstone inclusions (“pure”) from the XYC deposit contain 2.2 to 4.0 ppb Re and 93.4 to 123.4 ppt Os; pure pyrite from the DON deposit is significantly more enriched in Re and Os (34–37 ppb Re; 636.8–694.9 ppt Os). The ¹⁸⁷Re/¹⁸⁸Os values of pure pyrite separates from the XYC and DON deposits range from 137.6 to 197 and 182.1 to 201.4, respectively. Regression of all pure pyrite Re-Os data from both deposits yields an isochron age of 442 ± 14 Ma (MSWD = 7.4) and an initial ¹⁸⁷Os/¹⁸⁸Os (Os_i) value of 0.71 ± 0.07. The Re-Os age indicates that the early phase of pyrite precipitation (and by inference, sphalerite and galena) occurred during the early Silurian, consistent with biostratigraphic ages of the host rocks. The Os_i value of ∼0.8 for earliest Silurian seawater recorded from organic-rich shale in the basal Silurian Global Stratotype Section and Point (GSSP) at Dobs Linn, Scotland is very similar to that provided by the Howards Pass pyrite regression and hence suggests a hydrogenous (seawater) source of Os for the pyrite. Therefore, two possible sources of Os are (1) the Zn- and Pb-bearing hydrothermal fluid that leached Os from footwall sedimentary rocks, which were deposited in seawater, or (2) directly from seawater during precipitation of the pyrite, which suggests that the Os content of the hydrothermal fluid was minor relative to that of seawater.