Premise of the study: Polyploidy is a major genetic driver of ecological and evolutionary processes in plants, yet its effects on plant interactions with mutualistic microbes remain unresolved. The legume-rhizobium symbiosis regulates global nutrient cycles and plays a role in the diversification of legume taxa. In this mutualism, rhizobia bacteria fix nitrogen in exchange for carbon provided by legume hosts. This exchange occurs inside root nodules, which house bacterial cells and represent the interface of legume-rhizobial interactions. Although polyploidy may directly impact the legume-rhizobium mutualism, no studies have explored how it alters the internal structure of nodules. Methods: We created synthetic autotetraploids using Medicago sativa subsp. caerulea. Neotetraploid plants and their diploid progenitors were singly inoculated with two strains of rhizobia, Sinorhizobium meliloti and S. medicae. Confocal microscopy was used to quantify internal traits of nodules produced by diploid and neotetraploid plants. Key Results: Autotetraploid plants produced larger nodules with larger nitrogen fixation zones than diploids across both strains of rhizobia, although significance of this difference was limited by power. Neotetraploid M. sativa subsp. caerulea plants also produced symbiosomes that were significantly larger, nearly twice the size, than those present in diploids. Conclusions: This study sheds light on how polyploidy directly affects a plant-bacterial mutualism and uncovers novel mechanisms. Changes in plant-microbe interactions that directly result from polyploidy likely contribute to the increased ability of polyploid legumes to establish in diverse environments.