Infant botulism, the most common form of botulism in the United States, occurs when botulinum toxin-producing clostridia colonize the infant's gut and produce botulinum neurotoxin (BoNT). This condition is treated with botulinum antitoxin and supportive care; antibiotics are avoided due to the risk of worsening symptoms by promoting toxin release. Consequently, antimicrobial resistance genes (ARGs) are rarely investigated in Clostridium botulinum strains. During 2020 through 2023, 226 C. botulinum isolates associated with infant botulism were sequenced in our laboratory using whole-genome sequencing (WGS) technologies. In silico analysis of the WGS data revealed that 15% of these isolates (n = 36) carried a vatD gene variant, which is associated with resistance to streptogramin A antibiotics. In this study, we describe the molecular characterization of 36 isolates that harbor the vatD gene. To the best of our knowledge, this is the first report of the detection of the vatD gene in the chromosome of C. botulinum recovered from infants in the United States.IMPORTANCEThe continuous expansion of whole-genome sequencing (WGS) technologies, combined with refined data analysis tools, has enabled an in-depth analysis of pathogens, allowing a thorough characterization and comprehension of the genomic diversity of C. botulinum. In the present study, we demonstrate how the implementation of WGS into laboratory surveillance workflow allowed the detection of an antimicrobial resistance gene, the vatD gene, in the chromosome of C. botulinum strains recovered from infants. To the best of our knowledge, this is the first study to report the detection of the vatD gene in non-enterococci isolates recovered from clinical samples in the United States.