Streptococcus pneumoniae is commonly carried asymptomatically in the human nasopharynx, but it also causes serious and invasive diseases such as pneumonia, bacteremia, and meningitis, as well as less serious but highly prevalent infections such as otitis media. We have previously shown that closely related pneumococci (of the same capsular serotype and multilocus sequence type [ST]) can display distinct pathogenic profiles in mice that correlate with clinical isolation site (e.g., blood versus ear), suggesting stable niche adaptation within a clonal lineage. This has provided an opportunity to identify determinants of disease tropism. Genomic analysis identified 17 and 27 single nucleotide polymorphisms (SNPs) or insertions/deletions in protein coding sequences between blood and ear isolates of serotype 14 ST15 and serotype 3 ST180, respectively. SNPs in raffinose uptake and utilization genes (rafR or rafK) were detected in both serotypes/lineages. Ear isolates were consistently defective in growth in media containing raffinose as the sole carbon source, as well as in expression of raffinose pathway genes aga, rafG, and rafK, relative to their serotype/ST-matched blood isolates. Similar differences were also seen between serotype 23F ST81 blood and ear isolates. Analysis of rafR allelic exchange mutants of the serotype 14 ST15 blood and ear isolates demonstrated that the SNP in rafR was entirely responsible for their distinct in vitro phenotypes and was also the determinant of differential tropism for the lungs versus ear and brain in a mouse intranasal challenge model. These data suggest that the ability of pneumococci to utilize raffinose determines the nature of disease.IMPORTANCES. pneumoniae is a component of the commensal nasopharyngeal microflora of humans, but from this reservoir, it can progress to localized or invasive disease with a frequency that translates into massive global morbidity and mortality. However, the factors that govern the switch from commensal to pathogen, as well as those that determine disease tropism, are poorly understood. Here we show that capacity to utilize raffinose can determine the nature of the disease caused by a given pneumococcal strain. Moreover, our findings provide an interesting example of convergent evolution, whereby pneumococci belonging to two unrelated serotypes/lineages exhibit SNPs in separate genes affecting raffinose uptake and utilization that correlate with distinct pathogenic profiles in vivo This further underscores the critical role of differential carbohydrate metabolism in the pathogenesis of localized versus invasive pneumococcal disease. Copyright © 2019 Minhas et al.