The neurotoxin formed by Clostridium botulinum Group II is a major cause of foodborne botulism, a deadly intoxication. This study aims to understand the genetic diversity and spread of C. botulinum Group II strains and their neurotoxin genes. A comparative genomic study has been conducted with 208 highly diverse C. botulinum Group II strains (180 newly sequenced strains isolated from 16 countries over 80 years, 28 sequences from Genbank). Strains possessed a single type B, E, or F neurotoxin gene or were closely related strains with no neurotoxin gene. Botulinum neurotoxin subtype variants (including novel variants) with a unique amino acid sequence were identified. Core genome single-nucleotide polymorphism (SNP) analysis identified two major lineages-one with type E strains, and the second dominated by subtype B4 strains with subtype F6 strains. This study revealed novel details of population structure/diversity and established relationships between whole-genome lineage, botulinum neurotoxin subtype variant, association with foodborne botulism, epidemiology, and geographical source. Additionally, the genome sequences represent a valuable resource for the research community (e.g., understanding evolution of C. botulinum and its neurotoxin genes, dissecting key aspects of C. botulinum Group II biology). This may contribute to improved risk assessments and the prevention of foodborne botulism.