To estimate the invasive disease potential of Streptococcus pneumoniae serotypes, invasive isolates (n=138) were compared with nasopharyngeal isolates (n=153) from children under 6 years of age in the Czech Republic. Odds ratios (ORs) based on a comparison of the distribution of S. pneumoniae serotypes amongst invasive and carriage isolates were calculated for individual serotypes and 172 strains were characterized using multilocus sequence typing. The ORs of serotypes 9V and 14 were significantly greater than 1, suggesting an association with invasive disease, while serotypes 6A and 23F were significantly associated with carriage (ORs less than 1). A single predominant clone with high invasive disease potential was found in each of the 9V, 7F, 14 and 1 serotypes while carriage-associated serotypes were highly heterogeneous.