Genetic variants of Clostridium difficile have been reported with increasing frequency, but their true incidence is unknown. C. difficile strains have been classified into variant toxinotypes according to variations in the pathogenicity locus encoding the major virulence factors, toxins A and B. Some strains produce an additional toxin, binary toxin CDT. This survey of clinical isolates (153) from patients in a single hospital set out to ascertain the distribution of variant toxinotypes and strains possessing binary toxin genes. A PCR-RFLP-based method of toxinotyping identified 123 (80.4 %) isolates as toxinotype 0, 13 (8.5 %) strains as non-toxigenic and 17 (11.1 %) as belonging to variant toxinotypes. Binary toxin genes were amplified by PCR in nine strains (5.8 %), all of which were variant toxinotypes. Toxin variants of C. difficile are pathogenic and commonly isolated and need to be considered when evaluating new diagnostic testing strategies for C. difficile disease.