During early infancy asymptomatic intestinal colonization by Clostridium difficile is frequent. To update information on infant colonization prevalence and to characterize infant strains, in terms of their virulence factors and their phylogenetic diversity, a prospective screening of C. difficile in the stools of infants 0 to 2 years old was conducted at Jean Verdier Hospital (Hopital Jean Verdier) over an 18 month period. C. difficile was screened by toxigenic culture, and molecular characterization was performed by PCR-ribotyping and multilocus sequence typing (MLST). The overall C. difficile colonization prevalence was 33.7% (99/294). The colonization rate by a toxigenic strain was 7.1 % (21/294). Community-acquired C. difficile accounted for 66.7% (66/99) of cases. Molecular typing was performed on 90 isolates from Jean Verdier Hospital and 8 additional isolates from another hospital in Versailles (Centre Hospitalier de Versailles). Among these isolates, 23 were toxigenic (21 tcdA(+) /tcdB(+) and 2 tcdA(-)/tcdB(+)). All the isolates were negative for the binary toxin genes. Seventeen PCR ribotypes (PRs) were identified, with five PRs accounting for 82.7% (81/98) of the isolates. MLST generated 15 different sequence types (STs). The predominant genotype, PRJV11-ST38 (33.7%), included only non-toxigenic strains. Toxigenic strains were distributed in eight genotypes. Neither PR027-ST3, nor PR078/126-ST49 were identified but some PRs/STs corresponded to well-known adult infectious strains. These results indicate that infants are widely colonized by non-toxigenic strains. However, toxigenic adult infectious strains circulate in asymptomatic infants even in the community; thus, infants may be a reservoir for adult infectious strains.