Digital dermatitis (DD) is a highly contagious and infectious disease in cattle which has a considerable negative economic impact worldwide, and adversely affects animal welfare. Members of the genus Treponema are the only bacterial agents for which there is consistent evidence of participation in DD lesions. In Chile, DD has been described since the 1990s, but only under a clinical approach. To date, the presence of the pathogenic agent has not been confirmed in Chile by any type of confirmatory microbiological diagnosis. The aim of the present study was to detect the presence of Treponema spp. DNA in lesions consistent with DD, in Chilean dairy cattle for the first time. We provide PCR confirmation of Treponema spp. in Chilean dairy cattle affected by DD. The high rate of positive results, as well as the proportion of the main Treponema species involved, is in line with what have been described in published studies elsewhere. Future herd control plans should benefit from the molecular detection of these pathogenic bacteria associated with DD.