Aims The aim of this study was to determine phenotypic and genotypic resistance, virulence and clonal relationship of aeromonads and related species isolated from Czech carp fisheries. Methods and Results Forty-nine isolates obtained from a total of 154 fish from three breeding facilities were species identified using matrix-assisted laser desorption/ionization time of flight and the sequencing of the rpoB housekeeping gene. Most Aeromonas isolates were identified as Aeromonas veronii (94%, n = 34). Susceptibility to six antibiotics (oxytetracycline, flumequine, florfenicol, sulphamethoxazole/trimethoprim, enrofloxacin and oxolinic acid) was tested using the disc diffusion method. The presence of resistance genes and virulence factors was verified by PCR and sequencing, and the clonal relationship was analysed using pulsed-field gel electrophoresis (PFGE). Phenotypic resistance to one or more antimicrobials was found in 32 isolates (65%, n = 49). Resistance to oxytetracycline was the most common (41%) and associated mainly with the presence of tet(E) gene, while the percentage of isolates resistant to florfenicol was low (2%). Isolates carried one to five of the tested virulence factors and showed high diversity of PFGE profiles. Conclusions Since the highest percentage of antimicrobial resistance in aeromonads was found for oxytetracycline and the lowest percentage for florfenicol, it is suggested that florfenicol could be an adequate treatment alternative in carp fisheries. Significance and Impact of the Study Increasing resistance of aeromonads to commonly used antimicrobials has become an emerging problem in fisheries. This study was conducted in relation to the practical needs to identify a suitable antibiotic as an alternative to oxytetracycline.