Three psychrotrophic bacteria, morpho-physiologically, identified as Bacillus subtilis MRLBA7, Bacillus licheniformis MRLBA8 and Bacillus megaterium MRLBA9 were isolated from -20°C freezer of the Microbiology Research Laboratory (MRL), Quaid-i-Azam University, Islamabad, Pakistan. These strains were able to grow aerobically at 6°C but not at 40°C except MRLBA8 that could grow at 48°C. None of the isolates showed inhibition of growth in the presence of glycerol. Isolate MRLBA7, bearing central spore, grew in the presence of 30% glycerol at 0°C after 48 h of incubation and showed maximum growth without glycerol at 25°C after 24 h. Isolate MRLBA8 showed growth in the presence of 50% glycerol at 4°C after 72 h of incubation and maximum growth was observed at 20°C in the absence ofglycerol. Isolate MRLBA9 showed growth at 6°C in the presence of 40% glycerol after 48 h of incubation and maximum growth was observed at 25°C in the absence of glycerol. Isolates were susceptible to antibiotics except Bacillus subtilis MRLBA7 that exhibited antibiotic resistance against penicillin and fosphomycin, Bacillus licheniformis MRLBA8 against aztreonam and fosphomycin, and Bacillus megaterium MRLBA9 against vancomycin and penicillin. The growth profile and biochemical characteristics of all the isolates were rather similar to that of mesophilic counterparts except adaptation to low temperature. These strains could be used as model microbial strains for characterization of food contaminants in freezers, to understand the mechanism of antibiotic resistanceinduced at low temperature and as a source of psychrotrophic enzymes.