Crop rotation is a common agricultural practice for soybean production to suppress soil-borne pathogens and improve production. Our research is exploring benefits from 3-yr rotation of soybean, corn and wheat compared with traditional 2-yr rotation of soybean and corn. The field experiments were established in 2013 at Northwest Agricultural Research Station (NWARS) and Western Agricultural Research Station (WARS) of the Ohio Agricultural Research and Development Center. The soybean seedling establishment, biomass and yield were collected from rotation fields. Since 2016, after one round of 3-yr rotation, NWARS soybean yield followed a pattern of yield increase. Specifically, in 2018, the 3-yr rotation soybean from NWARS had 34% greater seedling root biomass and yielded 5% more compared to the 2-yr rotation. However, in WARS, 3-yr rotation did not affect soybean production compared with 2-yr rotation. Thus, the 3-yr rotation effects on plant biomass and yield differed between locations. The research will continue to test NWARS and WARS soil quality and rhizosphere bacterial community from both soybean rotation fields. The correlations of the measured soil and microbial parameters with soybean production in 3-yr rotation will be analyzed. We predict that the 3-yr rotation will have more diversity of soil bacterial community compared with the 2-yr rotation, regardless of site, and soil quality is higher in soybean 3-yr rotation field compared with 2-yr rotation.