Background:Clostridioides difficile is a toxin-producing bacterium that is the foremost cause of healthcare-associated diarrhea in the United States. Recent epidemiologic and genomic evidence indicates that divergent C. difficile strains have varying propensities for transmission within healthcare settings. We investigated whether and how these differences are reflected in the genomic epidemiology of 2 common C. difficile strains—sequence type (ST) 1 (analogous to Ribotype 027) and ST2 (associated with Ribotypes 014/020)—across 3 geographically distinct US medical centers. Methods: Between 2011 and 2017, a convenience sample of ST1 and ST2 C. difficile clinical isolates were collected from 3 US sites: The University of Michigan Medical Center, Texas Medical Center Hospitals, and Memorial Sloan Kettering Cancer Center. Isolates underwent whole-genome sequencing and in silico multilocus sequence typing to verify strain types. Sequences were mapped to ST1 and ST2 reference genomes and single nucleotide variants (SNVs) were identified, filtered, and used to construct pairwise SNV distance matrices. A range of pairwise SNV distance thresholds were applied to assess genetic linkages consistent with recent transmission within ST1 compared to within ST2. Proportions of genetically linked isolates were compared using 2 tests. Results: We identified 200 ST1 and 188 ST2 isolates across the 3 collection sites. Overall, ST2 was more genetically diverse than ST1 (pairwise SNV distance range, 0–156 SNVs and 0–78 SNVs, respectively). ST2 isolates displayed significantly less evidence of recent transmission: 10 ST2 isolates (5.3%) were within 2 SNVs of another isolate compared to 88 (44%) ST1 isolates (P .001) (Fig. 1). As the SNV threshold increased to 5 and 10 SNVs, this trend was maintained (all P < .001). ST2 isolates were also more likely to be genetically linked to an isolate from a different collection site than ST1 isolates. Among isolates with genetic links to at least 1 other isolate at the 5 SNV and 10 SNV thresholds, 21 of 37 and 74 of 89 ST2 isolates (57%, 83%) were linked to an isolate from a different collection site, compared to 2 of 88 and 48 of 157 ST1 isolates (2% and 31%, respectively; both P < .001). Conclusions: Compared to C. difficile ST1 isolates, ST2 isolates displayed less evidence of recent healthcare transmission and were more likely to be genetically linked to isolates from divergent collection sites. Interpreting genetic linkages among C. difficile isolates requires an understanding of regional and strain-specific genetic diversity to avoid misattribution of genetic linkages to recent transmission.