The ratios of Rb to Cs contents were studied in five fish species from seven lakes located in the Patagonia Andean Range, Argentina in order to trace fish diet. The species studied were native velvet catfish (Diplomistes viedmensis) and creole perch (Percichthys trucha), and exotic brown trout (Salmo trutta), rainbow trout (Oncorhynchus mykiss), and brook trout (Salvenilus fontinalis). Rainbow trout specimens from two farms were also studied, as well as fish food items and native mussels (Diplodon chilensis). Rb and Cs concentrations were determined by instrumental neutron activation analysis. A positive correlation of Cs concentration in the muscle of velvet catfish with fish length was observed, probably associated with the long biological half-life of this element in this species, whereas the Rb concentration remained constant, hence inhibiting the use of Rb-Cs ratios as a tracer in this case. Seasonal variations observed for rainbow trout and Cs concentration background bias in one of the lakes studied are also a limiting factor to the use of Rb-Cs ratios as a diet tracer. Rb-Cs ratios allowed clear differentiation of rainbow trout raised in farms from the natural specimens that lived in the same environment, in agreement with Rb-Cs ratios determined in both diets. Rb-Cs ratios in rainbow trout showed significant differences between Rivadavia and Futalaufquen lakes compared to Moreno and Nahuel Huapi lakes, which could be associated with a higher participation of plankton in the diet in the first case. No relevant variations in Rb-Cs ratios of brown trout were observed, probably because of the similarity in the diet.