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Most arable lands in the world are quite low in available phosphorus (P), becoming an important factor restricting crop yield. However, the genotypic difference of low-P tolerance in barley is rarely understood at molecular level. In this study, low-P responses of three barley genotypes were explored by physiological, transcriptomic and metabolomic analysis. The results showed that low-P-tolerant genotypes Zaoaibai (cultivar) and X130 (Tibetan wild accession) showed less growth inhibition compared to low-P-sensitive Salooni2 under low-P stress. Omics analysis showed that many genes involved in Pi acquisition and transport were more upregulated in Zaoaibai, which had relatively higher concentrations of shoot P and glucose-6-phosphate, indicating that enhancement of P acquisition, P translocation and P availability contributed to low-P tolerance in Zaoaibai. On the other hand, the genes and metabolites involved in biosynthesis of Pi-free lipids were highly expressed and more abundant in X130, suggesting that remodeling of membrane lipids by replacing phospholipids with Pi-free lipids is an important strategy for X130 in its adaptation to low-P stress. The divergent evolution of low-P tolerance in Zaoaibai and X130 could be driven by diverse ecological factors in Eastern China (low soil P) and Tibet Plateau (high soil P and low temperature), respectively. • Zaoaibai and X130 showed less growth inhibition compared to Salooni2 under low-P stress. • Enhancement of P acquisition, P translocation and P availability contributed to low-P tolerance in a cultivated genotype Zaoaibai. • Remodeling of membrane lipids by replacing phospholipids with Pi-free lipids is an important strategy for a Tibetan wild accession X130 in adaptation to low-P stress. • Divergent evolution of low-P tolerance in Zaoaibai and X130 may be driven by ecological factors in Eastern China (low soil P) and Tibet Plateau (high soil P and low temperature), respectively. [ABSTRACT FROM AUTHOR]