Analyses of the sulfur (S) and oxygen (O) stable isotope ratios (δ34SSO4 and δ18OSO4) of dissolved sulfate in streams and groundwater in Yukon Territory (YT, Canada) indicates that the dominant source of this major ion is oxidation of sulfide minerals. In these streams, sulfate has a large range in δ34SSO4 values (−19 to +10‰), which is consistent with the lower portion of the documented range for δ34S of sulfides in bedrock in YT (∼ −35 to +55‰). Furthermore, the large majority of the δ18OSO4 data plot within the expected field for sulfide oxidation in cross-plots of δ18OSO4 versus the O isotope ratios of ambient water (δ18OH2O). δ18OSO4 values plotting above that field were likely affected by microbial sulfate reduction, a process that enriches residual sulfate in both 34S and 18O. The stable isotope data indicate that dissolution of marine-evaporite gypsum, which has higher δ34SSO4 and δ18OSO4 values compared to all stream and groundwater samples, is a minor to negligible source of sulfate in the YT streams. Association of sulfate with other solutes indicates release of various metals from sulfide minerals, and suggests dominance of silicate weathering in response to oxidation of the sulfides. Variations in sulfate concentrations in YT streams are largely related to geology, while terrain and climate, including extent of permafrost, are also important factors. Long-term monitoring of sulfate concentrations in YT streams indicates increasing concentrations over time, in both streams impacted by mining, and in streams that have not been affected much by historical in-catchment industrial activities. Increases were largest in streams in northern and central-south YT in the presence of thawing continuous to discontinuous permafrost. Display omitted •Sulfate concentrations in Yukon Territory streams have increased over time.•Sulfide oxidation is the dominant source of sulfate in streams and groundwater.•Marine-evaporite gypsum is a minor to negligible source of stream sulfate.•Other solutes suggest silicate weathering as dominant response to sulfide oxidation.•Variations in sulfate concentrations in streams are largely related to geology.