Penicillium is the most common mould isolated in housing. Penicillium chrysogenum is the only species tested by prick test or serology for allergic patients. The American Institute of Medicine has accepted Penicillium as an aetiological agent of rhinitis in children and adults and as an asthma agent in children. However, few studies have identified Penicillium in housing to the species level (354 species). Phenotypic identification is difficult. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) should be an alternative. The aim of this study was (1) to identify the Penicillium species present in dwellings in Eastern France and (2) to evaluate the reliability of MALDI-TOF MS for identification, by comparing it to DNA sequencing and phenotypic identification.