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Objective: In-time diagnosis of Streptococcus pneumoniae (S. pneumonia) can play a significant role in decreasing morbidity and mortality rate. Applying molecular methods has gained popularity due to the existing limits of routine diagnostic methods. Examining the expression of different genes of this bacterium through different molecular methods suggests that lytA gene has a higher sensitivity and specificity in diagnosis of Streptococcus pneumoniae. The aim of this study was to evalutate lytA gene expression in diagnosis of invasive S. pneumonia in culture positive specimens by real-time polymerase chain reaction (PCR)., Materials and Methods: IIn this a descriptive study, All received specimens were isolated to identify S. pneumoniae. DNA was then extracted and after optimizing the test and determining the detection limit, samples were tested by real-time PCR using lytA gene primers., Results: Twenty seven isolates were diagnosed as S. pneumoniae. In all, the extracted DNA was positive in real-time method. The electrophoresis of the products also confirmed the presence of single product b along with the 53 base pair fragment. The detection limit of the test was less 6 colony forming unit (CFU)., Conclusion: Real-Time PCR seems to provide reliable and rapid results. We suggest that this test should be conducted on the preliminary isolated specimens, since applying various biochemical tests need one extra working day.