eThe incidence of Mycobacterium bovis, the causative agent of bovine tuberculosis, in cattle herds in the United Kingdom is increasing, resulting in substantial economic losses. The European badger (Meles meles) is implicated as a wildlife reservoir and is the subject of control measures aimed at reducing the incidence of infection in cattle populations. Understanding the epidemiology of M. bovis in badger populations is essential for directing control interventions and understanding disease spread; however, accurate diagnosis in live animals is challenging and currently uses invasive methods. Here we present a noninvasive diagnostic procedure and sampling regimen using field sampling of latrines and detection of M. bovis with quantitative PCR tests, the results of which strongly correlate with the results of immunoassays in the field at the social group level. This method allows M. bovis infections in badger populations to be monitored without trapping and provides additional information on the quantities of bacterial DNA shed. Therefore, our approach may provide valuable insights into the epidemiology of bovine tuberculosis in badger populations and inform disease control interventions.M ycobacterium bovis infection in wildlife is an issue of growing importance worldwide, with infections found in a range of species, including buffalo in Africa (1), wild boar in Spain (2), brushtail possums in New Zealand (3), and European badgers in the United Kingdom (4) and the Republic of Ireland (5). In the United Kingdom and the Republic of Ireland, badgers are involved in the transmission of tuberculosis (TB) to cattle (6-8). The incidence of M. bovis in cattle herds in the United Kingdom has been increasing for over 30 years (9), resulting in substantial economic losses (10). Once infected, badgers may intermittently shed M. bovis cells in sputum, feces, and urine (4), creating an environmental source of potential infection for other badgers and cattle (11,12). M. bovis DNA has been shown to survive outside the host for up to 21 months, and 16S rRNA has been detected in badger setts and latrines (13). In addition, studies have found a 2.5% positivity rate when culturing from badger feces (14), and M. bovis has been cultured from cattle feces several months after excretion (15). Furthermore, M. bovis that had persisted in soil for over 12 months was able to colonize mice (16). This indicates that at least a proportion of M. bovis cells shed in badger feces can remain viable in the environment. Monitoring M. bovis infections in badger populations is important for understanding the location and spread of disease and directing control efforts. TB control interventions targeted at badgers are currently based on culling, vaccination, and farm biosecurity (17).Accurate diagnosis of M. bovis infections in live animals is challenging yet essential in order to understand the epidemiology of the disease and its onward spread. Currently, infections in live badgers can be monitored through trapping and diagnosis with immunoassays (g...