Streptococcus pneumoniae is a major cause of sepsis, meningitis and respiratory disease worldwide. Pneumococcal conjugate vaccines (PCVs) have now been implemented in many countries worldwide, including Singapore. To evaluate the effectiveness of these vaccines, pneumococcal surveillance studies are required. Detailed and unified pneumococcal epidemiology data are currently scarce in South East Asia. Thus, we present data on invasive pneumococcal (IPD) isolates from Singapore that could assist in evaluating the effectiveness of pneumococcal vaccine in Singapore. One hundred and fifty-nine invasive pneumococcal disease isolates were received by the National Public Health Laboratory in Singapore between June 2009 and August 2010. Isolates were characterized using serotyping and multilocus sequence typing. Twenty-four different serotypes were found, the most common of which were 19A, 3, 7F, 23F, 6B, 14, 8 and 19F (in rank order). One hundred and two sequence types were observed, of which 38 were novel due to new alleles or new combinations of already existing alleles. Based on the Simpson’s Index of Diversity, serotypes 3, 6B and 19A were the most genetically diverse. Novel sequence types were more prevalent among conjugate vaccine serotypes 3, 19F and 23F and non-conjugate vaccine serotype 8, serogroup 15 and in non-typable isolates. We have demonstrated considerable genetic diversity among invasive pneumococci before and during the widespread use of conjugate vaccines in Singapore. Approximately half of all novel IPD clones identified in this study were non-conjugate vaccine serotypes. Although PCVs would target the most common serotypes, the high genetic diversity in non-vaccine serotypes would require further surveillance studies.