Ticks are known vectors of several viral, bacterial, and protozoal pathogens that cause disease in both humans and animals. While pathogen prevalence has been studied extensively in other portions of the United States, pathogen surveillance studies within tick populations in the central Appalachian region of Virginia is almost nonexistent. Two prominent species in this region are Ixodes scapularis (the blacklegged tick) and Amblyomma americanum (the lone star tick). In this study, we collected ticks biweekly from three habitat types (forest, urban, and pasture) across eight counties in southwest Virginia from June, 2019–November, 2020. Ixodes scapularis and A. americanum captures were screened for evidence of associated tick-borne pathogens. In this region, Borrelia burgdorferi sensu stricto (15.3% in nymphs and 37.6% in adults), Anaplasma phagocytophilum (1.9% in nymphs and 12.2% in adults), and Borrelia miyamotoi (2.97% in nymphs and 2.33% in adults) were detected in I. scapularis ticks. Aside from two previously reported Powassan-positive I. scapularis ticks from Floyd County, VA, no additional Powassan-positive ticks are reported here. No evidence of Ehrlichia chaffeensis, Heartland virus (HRTV), or Bourbon virus (BRBV) was detected in collected A. americanum. Detection and confirmation of multiple emerging tick-borne pathogens in this region raises an increased concern for public health risk, calling for heightened awareness of tick-borne pathogen transmission and increased tick surveillance in understudied areas.