An increasing proportion of children with acute otitis media due to Streptococcus pneumoniae have serotype 3 infections since licensure of the seven-valent pneumococcal conjugate vaccine. These serotype 3 strains are genetically related by molecular subtyping. During otitis media with effusion and recurrent otitis media, biofilms commonly develop. Pneumococcal in vitro biofilms are comprised of phase variants that differ in colony morphology. By using a representative strain of the mucoid serotype 3 clone, rough phase variants with a diverse array of mutations were detected in biofilms formed in vitro. Most phase variants had mutations in the cps3D gene, the first gene of the capsular operon. Eleven had single nucleotide polymorphisms (SNPs) in the cps3D gene, one had an SNP in the ؊10 promoter, and three had large deletions in the cps3D gene. Reversion to the mucoid phenotype was associated with reversion of the mutation in the cps3D gene. Unlike the phase variants detected in the nasopharynx, which have at least 20% of the parental amount of capsule, the in vitro biofilm-associated phase variants had <12% of the parental amount of capsule, as determined by capsule enzyme-linked immunosorbent assays. Using real-time reverse transcription-PCR, we determined that capsule expression in the phase variants was likely regulated at multiple levels. These in vitro phase variation data, which underscore the plasticity of the pneumococcus, need to be confirmed with in vivo analyses of the middle ear mucosa during otitis media.