Bovine digital dermatitis (DD) is a leading cause of lameness in dairy cattle throughout the world. Despite 35 years of research, the definitive cause of the disease process is still unknown. Previous studies have demonstrated that multiple bacterial species are associated with lesions, with spirochetes being the most reliably identified organism. This study utilized total DNA sequencing of 48 staged DD biopsy specimens collected during a 3-year longitudinal study of disease progression in dairy cattle. Over 175 million sequences were obtained and used to identify the bacterial species that were present in the biopsies. There was no evidence of a fungal or DNA viral causes. The bacterial communities present in the biopsy specimens was found to progress through a systematic series of changes that correlate with the novel visual lesion scoring system developed as part of this project. Although Treponema spp. predominated in the advanced lesions, they were in relatively low abundance in the newly described early lesions that are associated with the initiation of the disease process. The results of this study support the hypothesis that DD is a polybacterial disease process and provide unique insights into the temporal changes in bacterial populations throughout lesion development. These insights are expected to be critical in the development of new treatment strategies and the potential development of effective vaccines. In addition, the development and validation of a lesion scoring system will assist with determining the prognosis of a lesion.