In the present study, different geographical sources and sequence types (STs) of Clostridium difficile were preliminarily screened to investigate the distribution profiles of three core genes, VirB4, VirB6 and VirD4, of the type IV secretion system (T4SS). A total of 37 C. difficile strains from different sources were screened, inoculated and prepared for genome extraction. C. difficile toxins A and B were subjected to identification and multilocus sequence typing (MLST) analysis. The T4SS gene then underwent polymerase chain reaction amplification and sequencing analysis. Of the 37 strains, 25 were toxin A-and toxin B-positive, and 12 were toxin A-negative and toxin B-positive. MLST detected 11 strains with ST37, 10 with ST2, 6 with ST35, 7 with ST3, 1 with ST54, 1 with ST1 and 1 with ST119. The detection rates of VirB4, VirB6 and VirD4 were all 100% in colonies exhibiting T4SS. Single nucleotide polymorphisms (SNPs) were detected in a minority of strains. C. difficile strains with identical STs shared the same SNP loci for T4SS, and those with different STs had different SNP loci. The results of the present study may provide evidence for subsequent identification of T4SS distribution, epidemiological investigations, polymorphism analyses and research into the association between T4SS, cytotoxicity and enterotoxication in C. difficile.