ABSTRACT To determine whether the neurotoxin BoNT/B2 causing botulism in Spain is clonal, the genetic diversity and phylogenetic relationships of Clostridium botulinum from food-borne episodes and infant cases of the condition were explored. The botulinum toxin gene ( bont ) subtype, the variable region of the flagellin gene ( fla VR), and a seven-gene multi-locus sequence type were examined by sequencing 37 BoNT-positive cultures obtained over the period 2010 to 2022. Out of 37 botulism events, 16 food-borne episodes and 16 infant cases were associated with bont/b 2. Eight bont/b 2 alleles were detected [nucleotide distance range 0.0259–0.415%, Hunter and Gaston discrimination index (HGDI) 0.71]. The most common bont/b 2 allele corresponded to that of strain Prevot 25 NCASE and its single and double locus variations (87.5%). Four known fla VR types were identified (HGDI 0.79), along with one previously unknown ( fla VR-15). Sixteen sequence types (STs) (HGDI 0.89) were recorded including seven new STs (ST164–ST170; 10 new alleles) and five new STs (ST171–ST175; with new allele combinations) were also noted. Correlations among some STs and fla VR types were seen. Overall, the present results show that the combined analysis of bont/b 2- fla VR-ST at the nucleotide level could be used to track botulism events in Spain. The neurotoxin BoNT/B2 has largely been responsible for human botulism in Spain. The polymorphism analysis of bont/b2 , fla VR typing, and sequence type determinations, revealed a wide variety of clones to be responsible for human botulism, ruling out a common source of acquisition. IMPORTANCE Botulism, a potentially fatal disease, is classically characterized by a symmetrical descending flaccid paralysis, which if left untreated can lead to respiratory failure and death. Botulinum neurotoxin (BoNT), produced by certain species of Clostridium, is the most potent biological toxin known, and the direct cause of botulism. This study characterizes the acquisition in Spain of two forms of botulism, i.e., food-borne and infant botulism, which are largely caused by the main neurotoxin BoNT/B2. Polymorphism analysis of the bont/b2 gene, typing of the flagellin variable region sequence (flaVR), and multilocus sequence typing, were used to explore the genetic background of Clostridium botulinum group I. To our knowledge, this is the first phylogenetic and typing study of botulism undertaken in Spain.