Aerobic heterotrophic bacteria present in the surface water of three cold and nutrient-poor lakes in the Chilean Patagonia (Alto Reino, Las Dos Torres and Venus) were analysed for genetic similarity and metabolic diversity using 16S ribosomal DNA and the Biolog EcoPlate(TM) system, respectively. Bacterial fingerprints of water samples in enriched and non-enriched nutrient broth demonstrated a > 50% fingerprinting similarity between the lakes. Metabolic activity was also similar. However, the Biolog EcoPlate(TM) system carbon substrates revealed functional diversity. Lake Las Dos Torres showed the most fingerprinting similarity between enriched and non-enriched cold water samples. The amounts of living and viable bacteria were also higher in this lake's water sample, suggesting a predominance of facultative oligotrophic groups. DNA sequencing analysis demonstrated the presence of phylum Bacteroidetes in Lake Alto Reino; phyla Bacteroidetes and Gammaproteobacteria in Lake Las Dos Torres; and phyla Bacteroidetes, Alphaproteobacteria, and Gammaproteobacteria in Lake Venus. Although each lake had a unique bacterial community structure, the different bacterial groups may be performing similar metabolic functions, given the similarity in extreme environmental conditions.