Nile tilapia, Oreochromis niloticus (Linnaeus, 1758) is among the economically most important freshwater fish species in East Africa, and a major source of protein for local consumption. Human induced translocations of non-native stocks for aquaculture and fisheries have been found as a potential threat to the genetic diversity and integrity of local populations. In the present study, we investigate the genetic structure of O. niloticus from 16 waterbodies across Ethiopia using 37 microsatellite loci with SSR-GBAS techniques.