Background Crop rotation is an important management tactic that farmers use to manage crop production and reduce pests and diseases. Long-term crop rotations may select groups of microbes that form beneficial or pathogenic associations with the following crops, which could explain observed crop yield differences with different crop sequences. To test this hypothesis, we used two locations each with three long-term (14 year), replicated, crop rotation treatments: continuous corn ( Zea mays ) (CCC), corn/corn/soybean (SCC), and corn/soybean (CSC); both CSC and SCC had each phase present each year. In Year 15, we grew soybean ( Glycine max ) in each plot, so that soybean replaced corn in CCC and in the CSC phase where soybean grew in Year 14, and took data from soybeans following CCC (14 years of corn), SCC (two years of corn), CSC (one year of corn), and SCS (one year of soybean). Soybean yield and soil health indicators were measured, along with the bulk soil microbiome and soybean root-associated microbiome.Results Soybean yields were significantly higher following CCC than in the other three treatments at both locations. Soil protein as a soil health indicator was also higher following CCC than in the other treatments. Differential abundances of bacterial and fungal taxa were related to yield differences in a site-specific manner. Uncultured bacterial taxa in family JG30-KF-AS9 was enriched in the high-yielding CCC plots in Monmouth, whereas Microvirga , Rhodomicrobium , and Micromonosporaceae were enriched in the low-yielding SCS plots. Members of the fungal phylum Ascomycota were informative in explaining yield differences among treatments mostly as pathogens, but Tumularia , Pyrenochaetopsis and Schizothecium were enriched in the CCC plots, suggesting a role as either corn pathogens or beneficial fungal taxa for soybean. Multivariate analysis associated soil health indicators with the rotation regimes and some of the differentially abundant microbial taxa.Conclusions Our finding of associations between soil health indicators related to soil microbial populations and soybean yield following different cropping sequences has wide-ranging implications, opening the possibility of both monitoring and manipulating soil microbial populations as a way to improve crop yield potential.