Nonduplicate clinical isolates of Prevotella spp. recovered from patients hospitalized between 2003 and 2006 in two French tertiary-care teaching hospitals were investigated for their susceptibility to metronidazole and the presence of nim genes. Of the 188 strains tested, 3 isolates displayed reduced susceptibility to metronidazole after 48 h of incubation, while 27 additional isolates exhibited heterogeneous resistance after prolonged incubation; all 30 of the isolates were nim negative. Among the remaining 158 isolates, 7 nim-positive isolates were detected. All of these strains were identified as Prevotella baroniae by 16S rRNA gene sequence analysis and contained a new nim gene, named nimI, as determined by DNA sequence analysis. Chromosomal localization of this single-copy gene was demonstrated in all clinical isolates as well as in type strain P. baroniae DSM 16972 by using Southern hybridization. No known associated insertion sequence elements were detected upstream of the nimI gene in any of the nim-positive strains by PCR mapping. After prolonged exposure to metronidazole, stable resistant subpopulations could be selected in nimI-positive Prevotella isolates (n = 6) as well as in nim-negative Prevotella isolates (n = 6), irrespective of their initial susceptibility to this antibiotic. This study is the first description of a new nitroimidazole resistance gene in P. baroniae which seems to be silent and which might be intrinsic in this species. Moreover, our findings highlight the fact that high-level resistance to metronidazole may be easily induced in both nim-positive and nim-negative Prevotella sp. strains.