The environmental fates and consequences of intensive sulfur (S) applications to croplands are largely unknown. In this study, we used S stable isotopes to identify and trace agricultural S from field-to-watershed scales, an initial and timely step toward constraining the modern S cycle. We conducted our research within the Napa River Watershed, California, US, where vineyards receive frequent fungicidal S sprays. We measured soil and surface water sulfate concentrations ([SO4; 2−]) and stable isotopes (
δ; 34S–SO4; 2−), which we refer to in combination as the ‘S fingerprint’. We compared samples collected from vineyards and surrounding forests/grasslands, which receive background atmospheric and geologic S sources. Vineyard δ; 34S–SO4; 2− values were 9.9 ± 5.9‰ (median ± interquartile range), enriched by ∼10‰ relative to forests/grasslands (−0.28 ± 5.7‰). Vineyards also had roughly three-fold higher [SO4; 2−] than forests/grasslands (13.6 and 5.0 mg SO4; 2−–S l−1, respectively). Napa River δ; 34S–SO4; 2− values, reflecting the watershed scale, were similar to those from vineyards (10.5 ± 7.0‰), despite vineyard agriculture constituting only ∼11% of the watershed area. Combined, our results provide important evidence that agricultural S is traceable at field-to-watershed scales, a critical step toward determining the consequences of agricultural alterations to the modern S cycle.