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The symbiotic relationship between soybean and rhizobia facilitates nodulation and nitrogen fixation, providing a sustainable nutrient supply for increasing crop yields and reducing chemical fertilizer use. However, comparative studies on the conservation and strain-specificity of host gene expression regulated by different rhizobial strains remain limited. Here, we performed a comparative analysis between the previously isolated strain, Bradyrhizobium ottawaense Bott 59, and the model strain, Bradyrhizobium diazoefficiens USDA 110. Symbiotic phenotypes were evaluated after inoculation, and a root transcriptomic analysis was conducted at 3 dpi to assess early molecular responses. At 21 dpi, Bott 59-inoculated plants outperformed plants inoculated with USDA 110 in nodule number, nitrogenase activity, and biomass. Transcriptomic analysis revealed conserved host responses to both rhizobial strains, including NIN-mediated signaling, AON signaling, and the biosynthesis of phenylpropanoids and brassinosteroids. Further analysis revealed that Bott 59 specifically induced the expression of genes involved in isoflavonoid and flavonoid biosynthesis, including those encoding I2H, and HI4OMT. Moreover, Bott 59 triggered more pronounced transcriptional reprogramming in auxin, cytokinin, and jasmonic acid signaling pathways, along with differential expression of a broader set of transcription factor genes. Collectively, this study systematically unravels the conserved and strain-specific transcriptional regulatory events underlying host–rhizobium interactions. Our findings provide valuable theoretical insights and transcriptomic resources for further dissecting the molecular mechanisms of symbiotic nitrogen fixation (SNF), as well as for the targeted genetic improvement of crop nodulation and nitrogen fixation efficiency. Full article