Bovine digital dermatitis (BDD) is a contagious foot disease with worldwide occurrence in dairy cattle. The disease causes lameness and reduced animal welfare as well as economic losses for the farmer. The aetiology is not fully established but associations have been made withTreponemaspp. Today, BDD diagnosis is mainly based on visual inspection of cattle feet, therefore this study aimed to develop a multiplex quantitative PCR (qPCR) assay targetingTreponema phagedenis,Treponema pedis, Treponema medium, and‘Treponema vincentii’to aid in diagnosis. The assay was tested for specificity on 53 bacterial strains andin silicoon 168Treponemaspp. genomes, representative of at least 24 species. In addition, 37 BDD biopsies were analysed and the results compared to another qPCR assay published during the study period, which we modified by combining into a multiplex qPCR. The qPCR developed herein had a detection limit of 10 copies of each target species per PCR reaction. Both qPCR assays showed 100% specificity when tested on bacterial strains, but the qPCR developed in this study detected 3.4% moreT. phagedenis-positive biopsies of lesion category M1-M4.1 than the modified assay. To conclude, the developed qPCR assay detectingT. phagedenis,T. pedis, T. medium, and‘T. vincentii’has high analytical sensitivity and specificity and provides a useful complementary tool for diagnosis and epidemiological studies of BDD. The assay could possibly also be used for contagious ovine digital dermatitis (CODD) as similar bacteriological profiles have been suggested for BDD and CODD, especially regarding certainTreponemaspp.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11259-023-10147-5.