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Background: Identifying regional types and evaluating the frequency of pneumococcal strains has become increasingly important especially in vaccination. The purpose of this study was the identification and frequency determination of our regional serotype and evaluation of the performance of recent type specific multiplex PCR for the diagnosis of streptococcus pneumonia serotypes., Methods: All isolated S. pneumonia from suspected patients in Tehran and Isfahan Hospitals from June to December of 2012 were evaluated. All specimens and their serotypes were identified through a conventional method and specific antisera. Serotype specific multiplex PCR was applied and ran in seven reactions consisting of 34 S. pneumonia primer pairs plus a primer pair as an internal control for this purpose., Results: Overall, 14 genotype specific serotypes (including two subtypes for 19 and 23) were detected and had identical results with stereotyping method except for serotype 28 and one of the identified serotype 23. The serotypes 19, 6 and 23 were dominant with the frequency of 51.8%. A cross reactivity was also observed between genotypes 1 and 9A/9V., Conclusion: Applied multiplex PCR format can be suitable and cost effective tool for identification of S. pneumonia serotypes.