Objective To investigate the distribution and antibiotic resistance of pathogens isolated from respiratory tract and provide the basis for effectively control of respiratory infections and reasonably use of antimicrobial agents. Methods The distribution of pathogens isolated from respiratory tract in our hospital was reviewed retrospectively from January 1, 2009 to December 31, 2009 according to CLSI 2009. Antibiotic resistance was analyzed with WHONET 5.4 software. Results A total of 3 032 strains of microorganisms were collected, of which gram-negative bacillus accounted for 74. 1%, gram-positive coccus 15.7%, and fungus 10.2%. The top 10 species of pathogens were K. pneumoniae (13.8%), A. baumannii (12.7%), S. aureus (12.1%), P. aeruginosa (11.7%), E. coli (7.0%), S. maltophilia (4.9%), C. albicans (4.8%), H. influenza (4.3%), S. pneumoniae (3.5%) and E. cloacae (3.1%). The prevalence of H. influenzae was high in outpatients. A. baumannii strains showed relatively lower resistance to cef-operazone-sulbactam (7. 3%), but about 39. 1% of these strains showed intermediate resistance. Not more than 40% of A. baumannii strains were susceptible to other antimicrobial agents tested. P. aeruginosa isolates were highly resistant. The resistance rate ranged from 12.1% to 89.4%. Only 60%-70% of H. influenzae and S. pneumoniae isolates were susceptible to penicillins. The strains isolated from children were more resistant than those from adults. The prevalence of ESBLs-producing strains was 52.6% and 88.6% in K. pneumoniae and E. coli. The prevalence of MRSA was 75.1%. Multi-drug resistant strains were most common in ICU and Department of Respiratory Diseases, followed by Pediatrics. Conclusions The microbial isolates in our hospital during 2009 are mainly opportunistic pathogens. About 7.8% are fastidious bacteria. Most strains were resistant to multiple antibiotics. It is suggested that clinicians should use antibiotics rationally according to the results of susceptibility testing.