Ammonia is a common pollutant in aquaculture system, and toxic to all aquatic animals. However, different aquatic animals exhibit diverse physiological responses to high-level ammonia exposure, potentially indicating their divergent resistance to ammonia stress. In this study, juveniles of three freshwater turtles (Mauremys reevesii, Pseudemys nelsoni and Trachemys scripta elegans) were exposed to different concentrations of ammonia (0, 0.3 and 3.0 mg/L) for 30 days, and their swimming, growth performance, gut microbiota, and hepatic metabolites were measured to evaluate the interspecific difference in physiological responses to ammonia stress. Despite no differences in swimming ability, growth rate, and gut microbial diversity, observable changes in microbial community composition and hepatic metabolite profiles were shown in ammonia-exposed turtles. A relatively higher abundance of potentially pathogenic bacteria was found in M. reevesii than in the other two species. Moreover, microbial compositions and metabolic responses differed significantly among the three species. M. reevesii was, out of the three tested species, the one in which exposure to ammonia had the greatest effect on changes in bacterial genera and hepatic metabolites. Conversely, only a few metabolites were significantly changed in T. scripta elegans. Integrating these findings, we speculated that native M. reevesii should be more vulnerable to ammonia stress compared to the invasive turtle species. Our results plausibly reflected divergent potential resistance to ammonia among these turtles, in view of differential physiological responses to ammonia exposure at environmentally relevant concentrations.