Scant attention has been paid to Lyme disease, Borrelia burgdorferi, Ixodes scapularis, or reservoirs in eastern North Dakota despite bordering high-risk counties in Minnesota. Recent reports of B. burgdorferi and I. scapularis in North Dakota, however, prompted a more detailed examination. Spirochetes cultured from the hearts of five rodents trapped in Grand Forks County, North Dakota were identified as B. burgdorferi s. l. through sequence analyses of 16S, 16S-ileT intergenic spacer region, flaB, ospA, ospC, and p66. OspC typing revealed the presence of groups A, B, E, F, L, and I. Two rodents were concurrently carrying multiple OspC types. Multilocus sequence typing suggested the eastern North Dakota strains are most closely related to those found in neighboring regions of the Upper Midwest and Canada. BALB/c mice were infected with B. burgdorferi isolate M3 (OspC group B) by needle inoculation or tick bite. Tibiotarsal joints and ear pinnae were culture positive and B. burgdorferi M3 was detected by qPCR in tibiotarsal joints, hearts, and ear pinnae of infected mice. Uninfected larval I. scapularis were able to acquire B. burgdorferi M3 from infected mice; M3 was maintained in I. scapularis during the molt from larvae to nymph; further, M3 was transmitted from infected I. scapularis nymphs to naïve mice as evidenced by cultures and qPCR analyses. These results demonstrate isolate M3 is capable of disseminated infection by both an artificial and natural route of infection. This study confirms the presence of unique (non-clonal) and infectious B. burgdorferi populations in eastern North Dakota.