Objective: Several molecular serotype prediction methods have been published in order to simplify the pneumococcal serotyping. The aim of this study is to develop a molecular serotype prediction technique alternative to other molecular approaches. Material and Methods: S. pneumoniae serotypes 1, 3, 5, 6A, 7F, 8, 9V, 11A, 14, 15B, 18C, 19A, 19F and 23B, that are the most frequently isolated serotypes from infections in Turkey, were included in the study. S. pneumoniae cpsA and cpsB genes that are involved in the processing, regulation and export of the capsular polysaccharides, were amplified by polymerase chain reacsion (PCR). Then, heteroduplex analysis was performed to the 1.8 kb PCR products obtained from all serotypes with constant serotype 1. This technique was then modified to real-time fluorometric nucleic acid detection system. Results: The PCR products obtained from serotypes 1, 6A, 14 and 19F showed only homoduplex bands while those from serotypes 3, 5, 7F, 8, 9V, 11A, 15B and 23B showed specific heteroduplex bands. Although serotypes 18C and 19A showed a specific heteroduplex banding pattern, they could not be differentiated from each other. When further heteroduplex analysis was performed to serotypes 1, 6A, 14 and 19F with serotype 5 as a constant component, serotype 1 and 6A could be differentiated from the others. Heteroduplex and homoduplex DNA strands could be distinguished by melting curve analysis in real-time fluorometric nucleic acid detection system. Conclusion: As a rapid and cost-effective method, real-time heteroduplex analysis may be an alternative to other molecular methods for serotype prediction of pneumococci.