The authors aimed to get insights into the population structure of non-(sero)typable pneumococci (NTPn), a specific group of natural atypical pneumococci whose identification is often difficult, and which has remained insufficiently studied. A total of 265 presumptive NTPn, isolated between 1997 and 2003 from the nasopharynx of children, were characterized. Strains were confirmed to be pneumococci on the basis of bile solubility, and PCR detection or Southern blotting hybridization of lytA and psaA, genes ubiquitous in this species. Multilocus sequence typing (MLST) was used to exclude two isolates that gave ambiguous results. Non-typability was confirmed by the Quellung reaction using Omniserum. A total of 213 isolates were considered to be true NTPn. The molecular analysis of the true NTPn by PFGE and MLST showed that this population was genetically diverse, although a dominant cluster, accounting for 66 % of the isolates, was identified. Antimicrobial resistance was observed in most genetic backgrounds, and multidrug resistance to penicillin, erythromycin, clindamycin, tetracycline and sulfamethoxazole-trimethoprim was associated with strains belonging to the dominant cluster. Comparison with PFGE fingerprints and sequence types of large collections of serotypable strains showed that the genetic backgrounds of all but two NTPn were different from those found in serotypable strains. In addition, we found that NTPn strains with similar genetic backgrounds to those identified in our study had been isolated from disease sources in other countries. These observations seem to indicate that NTPn have diverse genetic backgrounds and may have evolved as a distinct group of pneumococcal isolates.