Aims <p>Kernza® (<i>Thinopyrum intermedium)</i> is among the first perennial grain crops ever bred and has the potential to address challenges associated with annual agriculture, in part due to its robust microbial partnerships and soil health benefits. However, breeding programs generally select plants based on aboveground traits such as seed size, resulting in unintended consequences on belowground traits such as root exudation and microbial symbioses. This study aimed to investigate the impact of plant breeding on the rhizosphere environment and microbial community of the novel grain crop Kernza.</p> Methods <p>This study is the first to investigate a crop’s rhizosphere environment across breeding cycles, starting with a wild-type population. We collected rhizosphere soil from Kernza plants across nine cycles of selection for increased yield and harvestability, and analyzed labile organic matter pools, potential enzyme activities, phospholipid fatty acids, and DNA from bacteria and arbuscular mycorrhizal fungi.</p> Results <p>We found that plant breeding altered bacterial and arbuscular mycorrhizal fungal communities in the rhizosphere. Selection also reduced rhizosphere labile organic matter and microbial biomass, but not microbial activity, which instead increased across later breeding cycles.</p> Conclusions <p>Our results suggest that the Kernza breeding program created a less abundant but more active rhizosphere microbial community, potentially due to impacts on plants’ stress tolerance, resource allocation, or resource-use strategies.</p>