Aims: Evaluate methods for identification and typing of Stenotrophomonas maltophilia isolated from a pharmaceutical facility. Methods and results: From 270 S. maltophilia strains identified by VITEK ®2, 40 were selected and submitted to MALDI TOF-MS, 16S and 23S rRNA gene analysis, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR), and an antimicrobial susceptibility profile. 16S rRNA sequencing was able to identify 39 (97.5%) strains as Stenotrophomonas spp. and one (2.5%) as Luteimonas huabeiensis . MALDI TOF-MS identified 37 (92.5%) strains as S. maltophilia , and three (7.5%) were not identified. PCR targeting 23S rRNA yielded a positive result for 39 (97.5%) strains. However, after sequencing , t wo strains were identified as Stenotrophomonas rhizophila , showing false-positive re- sults. The confirmed S. maltophilia strains ( n = 37) showed 35 distinct ERIC-PCR profiles and exhibited sensitivity to minocycline and levofloxacin, and six (16.3%) sho w ed intermediate resistance to sulf ametho xaz ole-trimethoprim. Conclusion: Matrix-assisted laser desorption lonization-time of flight mass spectrometry (MALDI-TOF MS) was a satisfactory methodology for the identification of S. maltophilia , but expansion of the database is necessary for the identification of other species. 16S rDNA sequencing sho w ed lo w resolution f or Stenotrophomonas species differentiation. PCR targeting 23S rRNA could not differentiate S. maltophilia from S. rhizophila . ERIC-PCR was shown to be a useful tool for the microbial source tracking of S. maltophilia . Impact Statement Not all methodologies can correctly identify S. maltophilia strains isolated in pharmaceutical facility. In the present study, MALDI-TOF MS was the most reliable method for the identification of S. maltophilia . Studies that discuss identification and the resistance profile of Stenotrophomonas maltophilia isolated from pharmaceutical industry environ- ments are very scarce. This information is extremely relevant in understanding the contamination source and its persistence in se v eral samples of the production chain.