Despite the significant development of antibiotics, sepsis is still associated with high morbidity and mortality rates. The identification of pathologic organisms at an early stage of sepsis is critical to improve the outcome, but this is difficult to achieve with the conventional method of blood culture (BC). It has been demonstrated that the genes of pathogenic organisms surviving in neutrophils were detectable with in situ hybridization (ISH) and this method was useful for the accurate and rapid diagnosis of sepsis. In this study, we applied ISH to blood smears 60 patients with suspected sepsis. BC was also carried out using the same blood samples to investigate the diagnostic value of ISH. The number of positive results obtained by ISH was approximately four times higher than that obtained by BC (ISH, 25 [41.7%]; BC, 7 [11.7%]). The positive rate in the 21 patients given antibiotics was 61.9% by ISH (13 patients) and 4.7% by BC (1 patient). The antibiotic treatments targeting the organisms detected by either procedure showed a beneficial clinical outcome. Positive results by ISH were obtained earlier than those with BC (ISH, within 1 day; BC, several days). We conclude that ISH is a useful method for the rapid diagnosis of sepsis.