In this work, 84 isolates of aeromonads were isolated from water and clinical samples, identified, and characterized. Identification was based on routine phenotyping combined with multiplex PCR. In this study, multiplex PCR was retested and reevaluated and its identification key was enhanced by 17 newly described species and five subspecies. Identification score increased from 36 % (only phenotyping) to 90 % when supported with multiplex PCR. Further description of isolates included detection of eight virulence genes. These genes were overall present in 46 % (act), 2.4 % (ast), 80 % (alt), 40 % (ahh1), 20 % (asa1), 69 % (pla/lip/lipH3/alp-1), 69 % (ser), and 81 % (fla), and no significant differences between water and clinical isolates were found. Results of this work show that the proper combination of different approaches is necessary for final identification of Aeromonas spp. at the species level. Multiplex PCR was shown to have limits in final identification, specifically inability to distinguish four species pairs and one triplet as their gene profiles are identical. However, it seems to be rapid and easy to do method able to support routine biochemical identification in laboratories. Moreover, our results supported previous proposal of reclassification of "Aeromonas hydrophila subsp. dhakensis" and "Aeromonas aquariorum" as identical species.