Abstract: Objective To retrospectively analyze the distribution of strains isolated from department of pediatrics and their drug resistance in order to provide the evidence for rational use of antibiotics. Methods Sputum and urine samples, collected from patients admitted to department of pediatrics in our hospital from June 2011 to June 2012, were cultured. Strains were identified and the minimum bacteria-inhibiting level of antibiotics was measured. Results A total of 231 strains were isolated from 522 samples with a positive detection rate of 44.3%. Of the detected 129(55.8%) Gram-negative bacilli, Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae accounted for 37.9% (49/129), 30.2% (39/129), and 16.3% (21/129), respectively. Of the detected 85(36.8%) Gram-positive strains, Staphylococcus aureus, coagulase- negative staphylococci and fungi accounted for 74.1% (63/85), 15.3% (13/85), and 7.36% (17/231) respectively. Of the detected 17 fungi, Candida albicans accounted for 82.3% (14/17). The ESβL–releasing Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae accounted for 66.7% (26/39), 24.5% (12/49) and 11.5% (3/26), respectively. The methicillin- resistant Staphylococcu saureus (MRSA) and methicillin-resistant coagulase-negative Staphylococci (MRCNS) accounted for 31.7% (20/63) and 76.9% (10/13), respectively. No vancomycin- resistant Enterococci (VRE)and vancomycin- resistant Staphylococcu saureus (VRSA) were detected. Gram-negative bacteria, especially Escherichia coli and Klebsiella pneumoniae, were highly sensitive to carbapenems, aminoglycoside and piperacillin/tazobactam while lowly sensitive to ampicillin, piperacillin and cephalosporin. Staphylococcus was lowly sensitive to penicillin and macrolide. All the strains were sensitive to vancomycin, streptogramins and oxazolidine. The nosocomial infection rate in department of pediatric of our hospital was 1.88% and the 3 nosocomial infection strains were multi drug-resistant. Conclusion Respiratory infection is the dominant disease in department of pediatrics. The major drug-resistant bacteria are ESβL –releasing Enterobacteriaceae, MRSA, and MRCNS. The result of the study is of certain significance for the rational use of antibiotics.