For several decades, the Midwestern USA has been impacted by blacklegged tick (Ixodes scapularis) range expansion, which, as the main vector of the Lyme disease-causing bacterium Borrelia burgdorferi, is linked to a regional increase in Lyme disease incidence. Earlier studies of genetic differentiation of blacklegged ticks have not tested detailed hypotheses about range expansion in the Midwest, despite the importance of this topic to public health. We addressed this gap by investigating the origin and environmental factors that influenced blacklegged tick establishment and spread in the Midwestern region. By analyzing fine-scale spatial population genomic data, we find low genetic differentiation consistent with the known recent range expansion. However, within Wisconsin, blacklegged ticks have unique genetic ancestries that differ from other Midwestern regions, suggesting multiple origins. Our data provide evidence for blacklegged tick sources in northern Wisconsin contributing to the recent expansion. In addition, we find a distinctive mixture of ancestry along the Mississippi River in southwestern Wisconsin and in Indiana, which was previously not identified. The most recently invaded populations in Michigan exhibit sharp genetic divergence from Wisconsin and Indiana samples despite their proximity, warranting further examination of their genetic origin and expansion processes. Lastly, landscape factors contribute to significant reductions in gene flow, potentially limiting genetic exchange and disease transmission within Midwestern states. This new knowledge of blacklegged tick range expansion processes can improve vector surveillance, pest management, and public health related to tick-borne disease risks.