The levels of 27 selected pesticides and trophic biomagnification of DDT were investigated in biota samples of the Lake Ziway in the Rift valley region, Ethiopia. Pesticide residues were analyzed using gas chromatography coupled with mass spectrometry (GC MS). Carbon source and trophic position were calculated by using 13C and 15N stable isotopes, individually, and trophic magnification factors (TMFs) were inferred. Among pesticides analyzed, only DDT and its metabolites (o,pʹ-DDD, o,pʹ-DDE, p,pʹ-DDD, and p,pʹ-DDE) were quantified in biota samples. The most prominent metabolite was p,pʹ-DDE with mean concentration ranging from the 0.22-7.7ngg− 1 wet weight. Moreover, the ratio of DDT/DDD + DDE in all the biota samples was less than 1 signifying historical DDT application. The trophic magnification factor (TMF) for p,pʹ-DDE, and ΣDDT were 1.18 and 1.19 respectively. Regression of log [ΣDDT] vs TL (trophic level) among all biota species showed a significant correlation, indicating that DDTs are biomagnifying along with the food web of Lake Ziway. The concentrations of DDTs and other organochlorine pesticides found in biota from Lake Ziway were, in general, lower than studies found in previous studies carried out in the same lake.