The aim of this study was to establish a simple method for the rapid identification of Mycobacteria species by MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass spectrometry) using the Bruker MALDI-TOF Biotyper system (Bruker Daltonik, Bremen, Germany). A multicentre, prospective, and single blind study was performed in three European Hospitals, two Spanish and one UK hospital from May to August 2018. The BD BACTEC MGIT (Becton Dickinson, Berks, UK) liquid culture system was used in all three centres for the growth of Mycobacteria. When signal positive, tubes were removed from the analyser and in addition to standard laboratory procedures were subcultured on blood agar plates for MALDI-TOF analysis. Plates were incubated aerobically for 1 to 7 days at 37 °C and inspected every day. Once any growth was visible, it was transferred to the steel target plate, overlaid with 1 μl of neat formic acid and 1 μl HCCA matrix (alpha hydroxyl 4 cinnamic acid), and analysed in a Bruker Biotyper MALDI-TOF. Results given by MALDI-TOF were compared with the reference methods used for identification in the different centres. At two Spanish hospitals, identification by MALDI-TOF was only attempted on presumptive non-tuberculosis mycobacteria (NTM) and the results were initially compared with the results obtained by a commercial reverse hybridisation assay, GenoType CM/AS (Hain Lifescience, Tübingen, Germany). At the UK Hospital, identification of any presumptive mycobacteria was attempted and compared with the results obtained by whole genome sequencing (WGS). Overall in 142/167 (85%) of cases the identifications obtained were concordant; all Mycobacterium tuberculosis (MTB) isolates 43/43 (100%), 57/76 (75%) of the rapid growing nontuberculous mycobacteria (NTM), and 42/48 (85%) slow growing NTM tested were identified correctly. We report a new, easy, cheap and quick method for isolation and identification of Mycobacterium spp. without the need for additional steps or equipment and this method is in routine used in all three centres.