To explore micromorphological and physiological responses of two contrasting genotypes of centipedegrass exposed to high-level Al3+and identify the main strategies in response to aluminum (Al) toxicity. The present study performed a micromorphologically and physiologically based comparative investigation of two contrasting genotypes (Al-sensitive E006 and Al-tolerant E105) of centipedegrass successively exposed to a high concentration of Al. The two genotypes showed an obvious difference in root micromorphogenesis after Al treatment, which resulted mainly from the different Al accumulations, with E006 having more Al accumulation. Al toxicity caused abnormalities in root morphogenesis, reductions in photosynthetic performance and relative water content, and increases in electrolyte leakage, malondialdehyde content, and hydrogen peroxide (H2O2) levels in plants. These effects were more pronounced in E006 than in E105. Meanwhile, Al treatment stimulated substantial increases in proline content and enzyme activity as defense responses in E105. Moreover, the significant elevation of endogenous hormone and the differences in the extent of the increase in the hormones abscisic acid (ABA) and salicylic acid (SA) between genotypes implied that the two hormones might mediate the above defense responses under Al toxicity. There are genotypic differences in the changes caused by successive Al treatment. The morphological and physiological damages caused by successive Al exposure in the Al-tolerant genotype were significantly lighter than those in the Al-sensitive genotype, which could be attributed to ABA- and SA-mediated positive defense responses mainly involving the less amount of Al accumulation, and the better-increased osmolytes and elevated antioxidant enzymes activity.