Cropping systems with perennial forages and reduced fallow frequency generally increase soil organic carbon and thus subsequent soil health and crop yield. We evaluated the impact of prior cropping systems on subsequent yields and soil properties in a semiarid region by using crop yields as a bioassay of soil health following the termination of a 24-year crop rotation study in the Brown soil zone in Alberta. During 24 growing seasons from 1992 to 2015, the study included three fallow-containing rotations, two annual crop rotations that were cropped continuously, and perennial grass hay, each with two to six fertilizer treatments. During the bioassay period from 2016 through 2020, all plots in the study were uniformly cropped. Compared to unfertilized fallow wheat, soil organic C in the fall of 2015 was 54% higher after 24 years of fertilized grass and up to 14% higher following annual crops in rotations without fallow. The most notable impact of the previous cropping system on yield during the bioassay years was low yield following perennial grass in 2016 and 2018. Soil electrical conductivity measurements showed that subsoil salinity was elevated following perennial grass, demonstrating the importance of subsoil characteristics for healthy soils. Crop yields in the fifth year of the crop bioassay were 10%–20% greater due to reduced fallow frequency or increased crop diversity. The long-term impact of the cropping system on crop yield in this study depended on drought intensity due to counteracting changes in soil organic matter and subsoil salinity.