A multilocus sequence analysis (MLSA) scheme was developed for characterization of strains and species from the genus Achromobacter, which are increasingly recovered from patients with cystic fibrosis (CF). Five conserved housekeeping genes were selected for the MLSA, which was applied to a diverse collection of 77 strains originating from Europe, Asia, and South America and including type strains of the seven recognized Achromobacter species, six environmental strains, eight non-CF clinical strains, and 56 CF clinical strains. The discriminatory power of MLSA, based on 2,098 nucleotides (nt), was much superior to a 16S rRNA gene comparison based on 1,309 nt. Congruence was observed between single-gene trees and a concatenated gene tree. MLSA differentiated all seven current Achromobacter species and also demonstrated the presence of at least four novel potential species within the genus. CF isolates were predominantly Achromobacter xylosoxidans (64%), an undescribed Achromobacter species (18%), and Achromobacter ruhlandii (7%). A clone of Achromobacter, which has spread among patients from Danish CF centers in Aarhus and Copenhagen, was identified as Achromobacter ruhlandii. MLSA facilitates the specific identification of isolates of Achromobacter necessary for describing their role in clinical infections.