The mutually beneficial relationship between plants and their root microbiota is essential for plants to adapt to unfavorable environments. However, the molecular mechanism of wheat regulating the structure of root microbiome and the influence of distant hybridization on this process are poorly understood. In this study, we systematically compared the root transcriptome and microbiome between a saline-alkali tolerant wheat introgression line SR4 (derived from somatic hybridization between wheat and tall wheatgrass) and its parent wheat variety JN177. The results indicated that root microorganisms were key factor maintaining better homeostasis of the sodium and potassium ion contents in SR4 than in JN177 under saline-alkali stress. Through systematic comparisons, we identified SR4-specific root bacterial and fungal taxa under saline-alkali stress. Through a weighted gene correlation network analysis (WGCNA) combining microbiome and transcriptome data, key functional genes and pathways, which were strongly related to root bacteria and fungi with differential abundance between JN177 and SR4, were identified. These results suggest that somatic hybridization has altered the key genes regulating root microbiome in wheat, further improving the saline-alkali tolerance of wheat introgression line. These findings provide the key bacterial and fungal taxa and functional target genes for wheat root microbiome engineering under saline-alkali stress. Display omitted •The mechanism of wheat regulating root microbiome is poorly understood.•Root microbes are key for the Na+/K+ content difference between JN177 and SR4.•SR4 recruits beneficial microbes through gene regulation to improve stress resistance.•Hybridization altered genes regulating root microbes, improving wheat stress tolerance.