Streptococcus pneumoniae is a commensal human pathogen and the causative agent of invasive pneumococcal disease. Carriage of the pneumococcus in the nasopharynx of humans is thought to be mediated by biofilm formation. Isogenic populations of S. pneumoniae grown under biofilm conditions frequently give rise to morphological colony variants, including small colony variant (SCV) phenotypes. This work employs phenotypic characterisation and whole genome sequencing coupled with ultra-pure liquid chromatography mass spectrophotometry (UPLC/MSE) of biofilm-derived S. pneumoniae serotype 22F pneumococcal colony morphology variants to investigate the diversification during biofilm formation. Phenotypic profiling revealed that SCVs exhibit reduced growth rates, reduced capsule expression, altered metabolic profiles and increased biofilm formation compared to the parent strain. Whole genome sequencing of 12 SCVs from independent biofilm experiments revealed that all SCVs studied had mutations within the DNA-directed RNA polymerase delta subunit (RpoE). Mutations included four large-scale deletions ranging from 51-264 basepairs (bp), one insertion resulting in a coding frameshift and seven nonsense single nucleotide substitutions that result in a truncated gene product. UPLC/MSE of the SCVs revealed up-regulation of a common sub-set of stress-inducible proteins which are part of an interaction network consisting of the 60 kDa chaperonin, chaperone protein DnaK, cell division protein FtsZ and manganese superoxide dismutase. This work links mutations in the rpoE gene to SCV formation and enhanced biofilm development in S. pneumoniae, with important implications for colonisation, carriage and persistence of the organism. Furthermore, consistent mutation within the pneumococcal rpoE gene presents an unprecedented level of parallel evolution in pneumococcal biofilm development. This work has given insight into the genetic diversity which may arise during pneumococcal colonisation which in turn may help inform future drug and vaccine design.