AbstractWinter rapeseed (Brassica napus L.) has a higher S requirement than most currently grown cereals and legumes. The objectives of this study were to (i) determine the effect of S fertilization rates on winter rapeseed yields and oil composition, (ii) establish relationships between winter rapeseed yields and soil SO4‐S levels for three cultivars, and (iii) determine the optimum time and depth of soil sampling for S diagnosis. Field experiments were conducted on a S deficient Latahco silt loam (fine‐silty, mixed, frigid, argiaquic Xeric Argiaboll) and two S deficient Palouse silt loam (fine‐silty, mixed, mesic pachic Ultic Haploxeroll) soils for three growing seasons. Each factorial trial had three cultivars, three S rates, and four replications. Soil samples were collected from all plots to a depth of 120 cm and sectioned into six 15‐cm and one 30‐cm sample in October, April, and June for SO4‐S analysis. Winter rapeseed yield was correlated to soil SO4‐S at different sampling depths. Both ‘Bridger’ and ‘Cascade’ cultivars responded to S; however, no response was observed for ‘Dwarf Essex’. The most significant coefficient of determination values between soil SO4‐S and rapeseed yield (Bridger r = 0.82, P = 0.01; Cascade r = 0.79, P = 0.01) were obtained with a 75‐cm soil sample from the April sampling. By linear plateau regression, critical soil test SO4‐S levels for Bridger and Cascade were 45 and 51 kg SO4‐S ha−1, respectively. This research demonstrates the need to modify current soil sampling for S management in winter rapeseed production to reflect the need for an April sampling to a 75‐cm depth.