The presence of harmful algal blooms (HABs) and cyanotoxins in drinking water sources poses a great threat to human health. The current study employed molecular techniques to determine the occurrence of non-toxic and toxic cyanobacteria species in the Limpopo River basin based on the phylogenetic analyses of 16S rRNA gene. The bottom sediments samples were collected from selected rivers: Limpopo, Crocodile, Mokolo, Mogalakwena, Nzhelele, Lephalale, Sand Rivers (South Africa); Notwane (Botswana), Shashe River and Mzingwane River (Zimbabwe). The physical-chemical analysis of the bottom sediments showed the availability of nutrients, nitrates and phosphates, in excess of 0.5 mg/l for most of river sediments, alkaline pH and salinity in excess of 500 mg/l. The FlowCam showed the dominant cyanobacteria species identified from the samples were Microcystis species, followed by Cylindrospermopsis raciborskii, Phormidium and Planktothrix species and this was confirmed by molecular techniques. Nevertheless, two samples showed the amplification of cylindrospermopsin polyketide synthetase gene (S3 and S9) while two samples showed amplification for microcystin/nodularin synthetase gene (S8 and S13). Thus these findings may imply the presence of toxic cyanobacteria species in the river sediments. The presence of cyanobacteria may be hazardous to human because rural communities and farmers who abstract water from Limpopo river catchment for human consumption, livestock and wildlife watering and irrigation.