Background : Several Acinetobacter species live in different ecosystems such as soil, freshwater, wastewater, and solid wastes. In this study, we assessed the occurrence of A. baumannii and A. nosocomialis , which are the major two clinically important species of the genus Acinetobacter , in three freshwater resources (Great Fish, Keiskemma, and Tyhume rivers) in the Eastern Cape Province, South Africa over a one year sampling regime (April 2017 - March 2018). Presumptive Acinetobacter species were subjected to molecular identification by using Acinetobacter genus-specific primers targeting the recA gene. The confirmed Acinetobacter species were further delineated into A. baumannii and A. nosocomialis using species-specific primer sets. Similarly, virulence genes, namely; afa/draBC, epsA, fimH, OmpA, PAI, sfa/focDE , and traT in the two Acinetobacter species were also determined using molecular techniques. Result : A total of 1107 presumptive Acinetobacter isolates were recovered from the freshwater resources of which 844 was confirmed positive for the Acinetobacter genus. Of the 844 Acinetobacter isolates, 285 (77%), 219 (70.9%) and 340 (79%) were recovered from Great Fish, Keiskemma and Tyhume rivers respectively. Our finding revealed that 410 (48.58%) and 23 (2.7%) of the isolates were confirmed to be A. baumannii and A. nosocomalis , respectively. The presence of these clinically-important Acinetobacter species in the freshwater studied suggests possible contamination of the selected rivers and also that A. baumannii and A. nosocomialis can thrive in aquatic environments. Besides, 308 (75.12%) A. baumannii and 3 (13.04%) A. nosocomialis isolates exhibited one or more virulence genes out of the seven tested, whereas 102 (24.88%) and 20 (86.95%) of the A. baumannii and A. nosocomialis isolates did not harbour any virulence gene. Additionally, OmpA was the most prevalent (p<0.05) virulence gene in A. baumannii with 69 (45.10%), 52 (50.98%) and 77 (49.68%) isolates in Great Fish, Keiskamma and Tyhume rivers respectively. Conclusion : The occurrence of these pathogens in rivers which are consumed by humans and livestock, as well as being used for irrigation system constitutes a risk to public health. Keywords: Freshwater resources, Molecular characterisation, Acinetobacter species, virulence gene s.