Tick-borne diseases are an emerging public health threat in the United States, but surveillance is lacking in some regions. To advance current knowledge of the ecology of ticks and tick-borne diseases in South Dakota, we conducted a survey in the summer of 2019, focusing on the eastern counties of the state. We collected and identified 266 ticks and a subset were tested for the presence of Borrelia burgdorferi by polymerase chain reaction (PCR). Dermacentor variabilis, a ubiquitous species in the state, was the most commonly identified tick, present in all counties surveyed. However, we also identified 15 Amblyomma americanum from three different locations, providing the first evidence of established populations in the state and expanding the range of this species. In addition, we identified 22 Ixodes scapularis from five different locations, confirming a previous report of an established population in the state. Two adult I. scapularis from two different sites were found to harbor B. burgdorferi, including an individual from Lincoln County, suggesting the ongoing presence of the pathogen in tick populations in the state and representing its southwestern-most detection in the midwest United States. These findings provide important information for assessing and monitoring the public health risk from tick-borne diseases in an area where surveillance is lacking.