A focused oligonucleotide microarray featuring 62 probes targeting strain variable regions of the Clostridium botulinum strain ATCC 3502 genome sequence was developed to differentiate C. botulinum type A strains. The strain variable regions were selected from deletions identified among a panel of 10 type A strains compared to the strain ATCC 3502 genome sequence using high density comparative genomic hybridization microarrays. The focused microarray also featured specific probes for the detection of the neurotoxin genes of various serotypes (A-G), toxin gene cluster components (ha70 and orfX1), and fldB as a marker for proteolytic clostridia (Group I). Eight pairs of strains selected from separate type A botulism outbreaks were included in the 27 subtype A1-A4 strains examined in this study. Each outbreak related strain pair consisted of strains isolated from different sources (stool and food). Of the eight outbreak related strain pairs, six groups of strains with indistinguishable hybridization patterns were identified. Outbreak related strains were shown to have identical hybridization patterns. Strain pairs from three separate outbreaks involving strains harboring both the type A neurotoxin gene (bont/A) and an unexpressed type B neurotoxin gene (bont/B) shared the same probe hybridization profile. The focused microarray format provides a rapid approach for neurotoxin gene detection and preliminary determination of the relatedness of strains isolated from different sources.