The opportunistic human pathogen Achromobacter (Alcaligenes) xylosoxidans has been recovered with increasing frequency from respiratory tract culture of persons with cystic fibrosis (CF). However, confusion of this species with other closely related respiratory pathogens has limited studies to better elucidate its epidemiology, natural history, and pathogenic role in CF. Misidentification of A. xylosoxidans as Burkholderia cepacia complex is especially problematic and presents a challenge to effective infection control in CF. To address the problem of accurate identification of A. xylosoxidans, we developed a PCR assay based on a 16S ribosomal DNA sequence. In an analysis of 149 isolates that included 47 A. xylosoxidans and several related glucose-nonfermenting species recovered from CF sputum, the sensitivity and specificity of this PCR assay were determined to be 100 and 97%, respectively. The availability of this assay will enhance identification of A. xylosoxidans, thereby facilitating study of the pathogenic role of this species and improving infection control efforts in CF.