Streptococcus pneumoniae polysaccharide and protein-conjugate vaccines are available against the most commonly isolated pneumococcal serotypes. Ongoing surveillance of invasive pneumococcal disease is needed in order to monitor changes in distribution of serotypes. Based on previously published sequences of capsular polysaccharide synthesis (cps) gene clusters of 16 pneumococcal serotypes, a molecular capsular typing (MCT) system has been developed, based on a combination of partial cpsA-cpsB sequencing and serotype- or serogroup-specific PCR, targeting the genes wzy and wzx (except for serotype 3). In this study, 151 S. pneumoniae isolates of known serotype (representing 51 serotypes) and 276 recent clinical isolates were used to develop MCT and compare it with conventional serotyping (CS) (total 427 isolates). On the basis of 376 heterogeneity sites in the cpsA-cpsB region, 89 sequence types (ST) were identified, of which 76 corresponded to a single serotype and 11 contained two serotypes. The correct serotypes in two of the latter (10A-23F-g and 23F-23A) were identified using serotype 23F-specific PCR. Limited CS was required for 92 (22 %) isolates to distinguish between the two serotypes in the nine other mixed ST (6A-6B-g, 6A-6B-q, 15B-22F, 33F-33A, 17F-35B, 18B-18C, 13-20, 25F-38, 31-42). MCT is a specific, objective and practical method that can predict the serotype of most S. pneumoniae isolates; it will facilitate epidemiological studies. Further study of the relationship between MCT and CS is needed in order to improve our understanding of serotype differentiation and to improve MCT methods further.