The contribution of mixed infection in recurrent Clostridium difficile infection (CDI) episodes is not known. Among paired isolates from 52 patients, mixed infection due to >1 toxigenic strain of C. difficile was identified in 8% of first episodes. Among recurrences, relapse from 1 or both co-infecting strains was uncommon; it was detected in a single case each. Infect Control Hosp Epidemiol 2016;1481-1484.