The important human pathogen Streptococcus pneumoniae is known to be a genetically diverse species. We have used comparative genome hybridization (CGH) microarray analysis to investigate this diversity in a collection of clinical isolates including several capsule serotype 14 pneumococci, a dominant serotype among disease isolates. We have identified three new regions of diversity among pneumococcal isolates and, importantly, clearly demonstrate genetic differences between strains of the same multilocus sequence type (ST) and capsule serotype. CGH may therefore, under certain circumstances, prove to be a valuable tool to supplement current typing methods. Finally, we show that these clonal strains with the same serotype and ST behave differently in an animal model. Strains of the same ST and serotype therefore have important genetic and phenotypic differences.