Abstract: High alkalinity severely limits Penaeus vannamei aquaculture. To elucidate the physiological mechanisms underlying long-term tolerance to high alkalinity, alkalinity-sensitive and alkalinity-tolerant families of P. vannamei were cultured for 80 days under either normal conditions (alkalinity 0.5 mmol/L, pH 8.2) or high-alkalinity stress (alkalinity 10.0 mmol/L, pH 8.9). The survival rate and growth index of the two lines were measured at 30 d and 80 d, respectively. Hemolymph samples were collected only at the end of the 80 d experiment, and the differences in osmotic pressure regulation ability (OC), blood ammonia concentration (BAC) and blood pH value were compared and analyzed. As for growth performance, the SGR of tolerant families was significantly higher than that of sensitive families at two time points. On the physiological level, the analysis of two-factor variance showed that the main environmental effect had a very significant impact on BAC at 80 d (P<0.01). OC is significantly influenced by the main effects of family (P=0.038) and E(P=0.016), but the interaction between them is not significant (P=0.383). Correlation analysis showed that SGR was positively correlated with OC (r=0.64, P<0.05), while OC was negatively correlated with BAC (r = 0.77, P<0.01). Linear regression further showed that the SGR of P. vannamei increased significantly with the increase of OC (R²=0.41, P=0.025), while OC decreased significantly with the accumulation of BAC (R²=0.59, P=0.003 5). The study shows that the growth advantage of tolerant families under long-term high alkaline stress stems from their remarkable osmotic pressure homeostasis maintenance ability and efficient BAC regulation ability. Osmotic pressure regulation, homeostasis, and BAC can be used as effective candidate physiological markers for alkali-tolerant breeding of P. vannamei, and this study provides a physiological basis and breeding reference for efficient breeding of saline-alkaline shrimp.