We investigated whether multilocus variable-number tandem-repeat analysis (MLVA) typing could identify different subtypes of Clostridium difficile ribotype 027 within the same feces specimen. Five of 39 specimens yielded at least one isolate with an MLVA profile different (more than five summed tandem repeat differences) from that of other isolates in the same specimen, thereby potentially obscuring epidemiological links between C. difficile infection cases.