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Clostridioides difficile toxins (TcdA and TcdB) are major exotoxins responsible for C. difficile infection (CDI) associated diseases. The previously reported TcdB variants showed distinct biological features, immunoactivities, and potential pathogenicity in disease progression. Here, we performed global comparisons of amino acid sequences of both TcdA and TcdB from 3,269 C. difficile genomes and clustered them according to the evolutionary relatedness. We found that TcdB was much diverse and could be divided into eight subtypes, of which four were first described. Further analysis indicates that the tcdB gene undergoes accelerated evolution to maximize diversity. By tracing TcdB subtypes back to their original isolates, we found that the distribution of TcdB subtypes was not completely aligned with the phylogeny of C. difficile. These findings suggest that the tcdB genes not only frequently mutate, but also continuously transfer and exchange among C. difficile strains. Shen et al. compare the amino acid sequences of bacterial toxins TcdA and TcdB from 3,269 Clostridioides difficile genomes to identify four new TcdB subtypes. They find that TcdB was more diverse in amino acid sequence than TcdA. This study suggests that the tcdB genes not only frequently mutate, but they also continuously transfer and exchange among C. difficile strains. [ABSTRACT FROM AUTHOR]