In Clostridium difficile, resistance to the macrolide-lincosamide-streptogramin B group of antibiotics generally relies on erm(B) genes. In this study, we investigated elements with a genetic organization different from Tn5398, the mobilizable non-conjugative element identified in C. difficile strain 630. Our results suggested that the elements most frequently found in strains isolated during the European surveillance study in 2005 were related to Tn6194, the conjugative transposon recently detected in different C. difficile types, including PCR-ribotype 027. We characterized a Tn6194-like and a novel element rarely found in clinical isolates. A burden on the in vitro fitness of C. difficile was observed after the acquisition of these elements as well as of Tn5398.