The aim of this study was to investigate the relationship between antimicrobial tolerance and taxonomic diversity among the culturable oxytetracycline-resistant (Ot r) heterotrophic bacterial population in two Belgian aquatic sites receiving wastewater either from human medicine or from aquaculture. The study of Ot r heterotrophs and mesophilic Aeromonas spp. allowed comparison of tolerance data at the intergenus as well as at the intragenus level. In total, 354 independently obtained Ot r isolates were subjected to antimicrobial tolerance testing and identified by GLC analysis of their cellular fatty acid methyl esters (FAMEs), by API 20E profiling and/or by Fluorescent Amplified Fragment Length Polymorphism (FAFLP) DNA fingerprinting. In general, Ot r hospital heterotrophs displayed a higher frequency (84%) of ampicillin (Amp) tolerance compared to the Ot r heterotrophs from the freshwater fishfarm site (22%). FAME results indicated that this effect was linked to the predominance of intrinsically ampicillin-resistant Ot r Aeromonas strains over representatives of Acinetobacter and Escherichia coli within the hospital strain set. Among the Ot r mesophilic Aeromonas strain set, the global tolerance profiles of the two sites only differed in a higher number of kanamycin (Kan) -tolerant strains (43%) for hospital aeromonads in comparison with the fishfarm aeromonads (8%). To some extent, this finding was correlated with the specific presence of Aeromonas caviae DNA hybridisation group (HG) 4. Collectively, these results suggest that the profiles for Amp and Kan tolerance observed in both sites arose from taxonomic differences in the culturable Ot r bacterial population at the generic or subgeneric level. In addition, our identification data also revealed that Enterobacter sp., Stenotrophomonas maltophilia, and A. veronii biovar sobria HG8 may be considered potential indicator organisms to assess microbial tolerance in various compartments of the aquatic environment.