BACKGROUND: The aim of this study was to investigate the effectiveness of the mental rotation test (MRT) based on virtual reality (VR) in predicting pilots' spatial visualization ability (SVA). METHODS: Based on VR, 118 healthy pilots' SVA were evaluated by MRT. The pilot flight ability evaluation scale was used as the criterion of test validity. According to the scale score, pilots were divided into high, middle, or low spatial ability groups pursuant to the 27% allocation principle. Differences in reaction time (RT), correct rate (CR), and correct number per second (CNPS) of MRT between groups were compared. Correlations between scale scores and MRT scores were analyzed. RT, CR, and CNPS of MRT among different age groups and between genders were also compared. RESULTS: The RT of the high spatial ability group was remarkably slower than that of the low spatial ability group (363.4 ± 140.2 s, 458.1 ± 151.7 s). The CNPS of the high spatial ability group was dramatically higher than that of the low spatial ability group (0.111 ± 0.045 s, 0.086 ± 0.001 s). There were no significant differences in RT, CR, and CNPS between different genders. Pilots in the 29-35 yr old age group had considerably slower RT than those in the 22-28 yr old age group (330.8 ± 140.3 s, 417.2 ± 132.7 s). Pilots in the 29-35 yr old age group had conspicuously higher CNPS than pilots in the 22-28 yr old age group (0.119 ± 0.040 s, 0.096 ± 0.036 s). All pilots' scale scores were positively correlated with CNPS ( r = 0.254) and negatively correlated with RT ( r = -0.234). DISCUSSION: MRT based on VR has a good discrimination efficacy for SVA of pilots and is a good indicator for the SVA component measurement. Zhang M, Wang M, Feng H, Liu X, Zhai L, Xu X, Jin Z. Pilots' spatial visualization ability assessment based on virtual reality . Aerosp Med Hum Perform. 2023; 94(6):422-428.