The aim of the present study was to characterize the volatile metabolites produced by genotypically diverse strains of the Stenotrophomonas genus in order to evaluate their potential as biomarkers of lung infection by non-invasive breath analysis. Volatile organic compounds (VOCs) emitted from 15 clinical and five environmental strains belonging to different genogroups of Stenotrophomonas maltophilia (n = 18) and Stenotrophomonas rhizophila (n = 2) cultured in Mueller-Hinton Broth (MHB) liquid media were analysed by gas chromatography mass spectrometry (GC-MS) and selected ion flow tube mass spectrometry (SIFT-MS). Several VOCs were detected in high concentration, including ammonia, propanol, dimethyl disulphide propanol and dimethyl disulphide. The GC-MS measurements showed that all 15 clinical strains produced similar headspace VOCs compositions, and SIFT-MS quantification showed that the rates of production of the VOCs by the genotypically distinct strains were very similar. All in vitro cultures of both the Stenotrophomonas species were characterised by efficient production of two isomers of methyl butanol, which can be described by known biochemical pathways and which is absent in other pathogens, including Pseudomonas aeruginosa. These in-vitro data indicate that methyl butanol isomers may be exhaled breath biomarkers of S. maltophilia lung infection in patients with cystic fibrosis.