Otitis, pneumonia, and meningitis are tissue-based pneumococcal infections that can be associated with biofilms. The emergence of phenotypic rough variants, also known as acapsular small-colony variants, is essential for pneumococcal biofilm formation. These rough variants can increase nearly 100-fold in biofilms over time and can arise through single nucleotide polymorphisms (SNPs), deletions, or tandem duplications in the first gene of the capsular operon, cps3D. We detected a 100-fold increase in rifampin-resistant (Rif(r)) mutants in biofilms compared to planktonic cultures using a nonvaccine serotype 3 strain, which is causing an increasing number of cases of otitis in the 7-valent pneumococcal conjugate vaccine era. Since both rough variants and Rif(r) strains can arise through SNPs, they could emerge due to alteration of the mismatch repair (MMR) system. The Hex system, a pneumococcal MMR system, repairs mismatches during replication and transformation. In this study, no mutations were detected in the hexAB gene sequences among several rough variants with unique mutations in the cps3D gene. Within a hexA null mutant grown in broth, we detected only a 17.5-fold increase in rough variants compared to the wild-type parental strain. Taken together, these data suggest that mutations in the hex genes and modulation of hexA activity are unlikely to account for the generation of biofilm-derived rough variants.