This study investigates the trophic transfer of persistent organic pollutants (POPs: PCBs, PBDEs, OCPs and PFASs) in the subtropical aquatic ecosystem of the Olifants River Basin (South Africa) by means of trophic magnification factors (TMFs). Relative trophic levels were determined by stable isotope analysis. POP levels in surface water, sediment and biota were low. Only ∑DDTs levels in fish muscle (1, indicating biomagnification of all detected POPs. Calculated TMFs for PCBs were comparable to TMF values reported from the tropical Congo River basin and lower than TMFs from temperate and arctic regions. For p,p'-DDT, a higher TMF value was observed for the subtropical Olifants River during the winter low flow season than for the tropical Congo river. TMFs of DDTs from the present study were unexpectedly higher than TMFs from temperate and arctic aquatic food webs. The fish species in the aquatic ecosystem of the Olifants River can be consumed with a low risk for POP contamination.