Although Streptococcus pneumoniae is a leading cause of childhood disease in South East Asia, little has previously been reported regarding the epidemiology of invasive pneumococcal disease in Malaysia and very few studies have explored pneumococcal epidemiology using multilocus sequence typing (MLST). Here we describe serotype, multilocus sequence type (ST), and penicillin susceptibility of thirty pneumococcal invasive disease isolates received by the University of Malaya Medical Centre between February 2000 and January 2007 and relate this to the serotypes included in current pneumococcal conjugate vaccines. A high level of diversity was observed; fourteen serotypes and 26 sequence types (ST), (11 of which were not previously described) were detected from 30 isolates. Penicillin non-susceptible pneumococci accounted for 33% of isolates. The extent of molecular heterogeneity within carried and disease-causing Malaysian pneumococci remains unknown. Larger surveillance and epidemiological studies are now required in this region to provide robust evidence on which to base future vaccine policy.