Introduction: Streptococcus pneumoniae is a well known Gram positive diplococci. This bacterium has the second most important pathogen in cases of meningitis in children under 2 years and elderly too. Currently more than 90 pneumococcal serotypes have been identified based on their antigenic differences in the capsular polysaccharides. Because the factors affecting serotype variation are not well defined. The aim of this study was to determine the difference between in the types of Streptococcus pneumoniae before and after lyophilization. Materials and methods: In this experimental study 50 clinical isolates of Streptococcus pneumoniae were collected and reidentified. Quelling reaction test as one of the serotyping methods were carried out (based on SSI protocol- Statens Serum Institute protocol).The results of the first step was recorded and then samples were lyophilized. In the second step the lyophilized samples were been reserotyped again and the results was compared. Results: The results of this study showed that there is no difference was occurred between the serotypes before and after lyophilization. Therefore lyophilization of Streptococcus pneumoniae has not affect on serotype diversity. In addition, we found that the isolates were belonging to serotypes of 7, 2, 5, 1, 6, 8, 4, 20, 3, 17, 19, 14, 22 and 10. Only one isolate was nonserotypable. Discussion and Conclusion: The finding of this study indicated that lyophilization use for maintenance samples can not change the types of Streptococcus pneumoniae isolates. If we haven’t any preparation for serotyping of isolates, we can use lyophilization as a way for conserve of our samples. On the other hand, when the equipment and material are not available in some case condition, we would perform lyophilize the clinical isolate and do serotyping later. In this research, serotyping has been done based on immunologic reaction but for accurate recognition due to the effects of lyophilization on serotypes diversity, further study is necessary to determine existence or inexistence of eventually mutation.