We studied the clinical utility of in situ hybridization (Hybrisep) in diagnosing respiratory infection and/ or sepsis. Peripheral blood was taken from patients with respiratory infections and suspected sepsis for both routine blood culture and in situ hybridization, and focal samples including sputum, bronchoalveolar lavage, and central and thoracic catheter, were simultaneously examined for bacterial culture. Specimens numbered 46. The clinical diagnosis was 20 cases of septicemia, with 26 specimens diagnosed as respiratory infectious diseases including hospital-acquired pneumonia and pleurisy. Positive cases of in situ hybridization were seen in 19 specimens (41.3%) in all specimens, significantly higher than that in blood culture (17.4%). Respiratory infection showed a high positive rate in in situ hybridization. Interestingly, the bacterial pathogen detected by in situ hybridization was not always consistent with that taken from focal samples. Whether the pathogen isolated by in situ hybridization is accurate etiologically or diagnostically remains unknown, but our findings suggest a polymicrobial infection in patients with hospital-acquired respiratory infections. In situ hybridization thus provides important information on the etiological pathogen in different infectious diseases.