This study estimated the value of in situ PCR (ISPCR) in the detection of Haemophilus influenzae type b (Hib) DNA in paraffin-embedded lung tissues of a murine pneumonia model. ICR mice were infected with Hib solution intranasally. In study group A (n-20), physiological changes and the number of deaths were recorded for 7 consecutive days after infection. In study group B (n=10), blood samples and lung tissues were obtained from the infected mice on the brink of death. In both groups, portions of the lung tissue were cultured for Hib, while other portions were submitted for histopathological studies. Conventional PCR, PCR followed by Southern blotting and ISPCR were performed to detect Hib in paraffin-embedded lung tissues. In control group A, six mice were inoculated intranasally with the same concentration of heat-inactivated Hib solution. In control group B, six healthy mice served as a blank control. Both control groups were managed using the same methods as those used in the study groups. The white blood cell count of the mice in the study group increased (F=3.295, P<0.01), with a high neutrophil Count (F=0.127, P<0.05). In the histopathological study, various stages of pneumonia were found in the lung tissues of the infected mice examined by microscope; 80% of the mice had moderate or severe pneumonia. Cultures of lung tissues in the study groups were all positive for Hib, while no bacteria were found in the control groups. Hib was detected in only 4 of 30 samples (13.3%) of the study groups using conventional PCR, but in all 30 samples (100%) using both Southern blotting and ISPCR. All three methods did not detect Hib in the control groups. Because of its sensitivity and specificity and its ability to locate the micro-organism, ISPCR can be considered suitable for the detection of Hib in paraffin-embedded lung tissues.