ABSTRACT A pilot study was conducted to determine the genetic diversity of multiple colonies of pneumococci recovered from 37 nasopharyngeal (NP) samples of children. A total of 239 pneumococcal isolates (typically, six to eight colonies per sample) were typed by pulsed-field gel electrophoresis (PFGE). In most NP samples (89%) the multiple colonies shared common PFGE types and serotypes. However, four samples were heterogeneous (samples A through D): each contained two strains with different PFGE types, antibiotypes, and serotypes. Samples A and B each contained one strain of a vaccine capsular type and another expressing a non-vaccine type (according to the currently licensed seven-valent conjugate vaccine). In samples B and C the penicillin MIC for one strain was elevated and the other strain was susceptible. In each of the heterogeneous samples, one of the strains was a representative of an internationally disseminated clone. Samples A, C, and D contained strains which carried prophages that were inducible by mitomycin C and that could be visualized by electron microscopy. The comC gene allele (which encodes the competence-stimulating peptide) was the same in both strains found in each of samples A, B, and D. Carriage of multiple pneumococci with distinct properties should favor genetic exchange and provide a dynamic population structure for pneumococci in their ecological reservoir. Quantitative resolution of majority and minority components of the pneumococcal NP flora will be of importance for evaluation of the impact of intervention strategies such as vaccination or introduction of new antimicrobial agents.