Litopenaeus vannamei has a high commercial value and is the primary cultured shellfish species globally. In this study, we have compared the hemolymph physiological responses between two L. vannamei strains under acute hypoxia. The results showed that hemocyanin concentration (HC) of strain A6410 was significantly higher than strain Zhengda; Total hemocyte counts (THC) decreased significantly in both strains under hypoxic stress (p < 0.05). We also investigated the temporal and spatial variations of hypoxia inducible factors 1 (HIF-1) by qRT-PCR. The results showed that hypoxia for 12 h increased the expression levels of HIF-1α in tissues of muscle and gill from the two strains (p < 0.05). In the hepatopancreas, the expression levels of HIF-1 increased significantly in strain Zhengda and decreased significantly in strain A6410 (p < 0.05). No significant changes of HIF-1 expression were detected in the same tissues between the two strains under hypoxia for 6 h (p > 0.05), but in the gills and hepatopancreas under hypoxia for 12 h (p < 0.05). Additionally, the expression level of HIF-1 was higher in the strain Zhengda than A6410 in the same tissue under hypoxia for 12 h. It was indicated that the hypoxic tolerance of Litopenaeus vannamei was closely correlated with the expression level of HIF-1, and the higher expression level of HIF-1 to hypoxia, the lower tolerance to hypoxia in the early stage of hypoxia. These results can help to better understand the molecular mechanisms of hypoxic tolerance and speed up the selective breeding process of hypoxia tolerance in L. vannamei.