AimsThe aim of the study was to determine the prevalence of Mycobacterium bovis (the causative agent of bovine tuberculosis, bTB) in environmental matrices within a French region (Cote d'Or) affected by this zoonotic disease. Methods and ResultsWe report here the development and the use of molecular detection assays based on qPCR (double fluorescent dye-labelled probe) to monitor the occurrence of Mycobacterium tuberculosis complex (MTBC) or Myco.bovis in environmental samples collected in pastures where infected cattle and wildlife had been reported. Three qPCR assays targeting members of the MTBC (IS1561 and Rv3866 loci) or Myco.bovis (RD4 locus) were developed or refined from existing assays. These tools were validated using Myco.bovis spiked soil, water and faeces samples. Environmental samples were detected positive for the presence of MTBC strains and Myco.bovis in the environment of bTB-infected farms in the Cote d'Or region. ConclusionsThe development of molecular assays permitted testing of several types of environmental samples including spring water, sediment samples and soils from badger setts entrance located in the vicinity of these farms, which were repeatedly contaminated with Myco.bovis (up to 87x10(3) gene copies per gram of badger sett soil). For the first time, direct spoligotyping of soil DNA enabled identification of Myco.bovis genotypes from environmental matrices. Significance and Impact of the StudyAll together, these results suggest that Myco.bovis occurs at low levels in environmental matrices in Cote d'Or within the bTB-infected area. Drinking contaminated water or inhaling contaminated bioaerosols might explain cattle infection in some cases.