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Molecular signatures of <i>Plesiomonas shigelloides</i> strain specific to pathogenic and nonpathogenic variants are not well established till present. There is a need for intra-species barcoding of <i>P. shigelloides</i> to aid infection control. This study aims at characterizing and assessing intra-species diversity and abundance of <i>P. shigelloides</i> isolated from three freshwaters in the Eastern Cape Province. The study used a Plesiomonas-specific PCR to characterize the isolates. Intra-species (dis)similarities were assessed using ERIC-PCR and (GTG)5-PCR techniques. The DNA fingerprints produced were electrophoresed, digitized, and documented via computer-assisted pattern analysis. The fingerprints were analyzed using neighbor-joining clustering (NJC) based on Euclidean similarity index. Results revealed 80%, 83.64%, and 80% of the water samples from Tyhume, Kat, and Kubusie rivers, respectively, positive for <i>P. shigelloides</i> isolation. The prevalence of <i>P. shigelloides</i> from sites ranged from 13.5% to 88.9%. NJC delineated 48 isolates to 8 clades (ERIC-fingerprints) and 34 isolates into 7 clades ((GTG)5-fingerprints). The relative abundance of unique strains ranged from 6.3% to 22.9% via the two methods. Both fingerprinting approaches have strain-differentiating potential for <i>P. shigelloides</i>, however ERIC-PCR possessed higher resolution (<b>D</b> = 37.46) advantage over (GTG)5-PCR (<b>D</b> = 29.64). In conclusion, the study achieved intra-species diversity and abundance of <i>P. shigelloides</i> from aquatic milieu and provide further opportunity for intra-species-specific barcoding.