Two approaches based on intact cell matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (IC-MALDI-ToF MS) have been evaluated in order to discriminate and identify nine former Burkholderia cepacia complex (Bcc) species, Burkholderia contaminans belonging to the novel Taxon K, Burkholderia gladioli, and the most relevant non-fermentative (NF) Gram-negative rods recovered from cystic fibrosis (CF) sputum cultures. In total, 146 clinical isolates and 26 reference strains were analysed. IC mass spectra were obtained with high reproducibility applying a recently developed inactivation protocol which is based on the extraction of microbial proteins by trifluoroacetic acid (TFA). In a first approach, spectral analysis was carried out by means of a gel-view representation of mass spectra, which turned out to be useful to recognize specific identifying biomarker proteins (SIBPs). A series of prominent mass peaks, mainly assigned to constitutively expressed proteins, were selected as SIBPs for identifications at the genus and species level. Two distinctive mass peaks present in B. contaminans spectra (7501 and 7900 Da) were proposed as SIBPs for the identification of this novel species. A second approach of spectral analysis based on data reduction, feature selection and subsequent hierarchical cluster analysis was used to obtain an objective discrimination of all species analysed. Both complementary modalities of analyzing complex IC-MALDI-ToF MS data open the path towards a rapid, accurate and objective means of routine clinical microbiology diagnosis of pathogens from sputum samples of CF patients.