The lone star tick, Amblyomma americanum, is an important disease vector and the most frequent tick found attached to humans in the eastern United States. The lone star tick has recently experienced a rapid range expansion into the Northeast and Midwest, but despite this emerging infectious threat to wildlife, livestock, and human health, little is known about the genetic causes and consequences of the geographic expansion. In the first population genomic analysis of any tick species, we characterize the genetic diversity and population structure of A. americanum across its current geographic range, which has recently expanded. Using a high-throughput genotyping-by-sequencing approach, we discovered more than 8,000 single nucleotide polymorphisms in 90 ticks from five locations. Surprisingly, newly established populations in New York (NY) and Oklahoma (OK) are as diverse as historic range populations in North and South Carolina. However, substantial population structure occurs among regions, such that new populations in NY and OK are genetically distinct from historic range populations and from one another. Ticks from a laboratory colony are genetically distinct from wild populations, underscoring the need to account for natural variation when conducting transmission or immunological studies, many of which utilize laboratory-reared ticks. An FST-outlier analysis comparing a recently established population to a long-standing population detected numerous outlier sites, compatible with positive and balancing selection, highlighting the potential for adaptation during the range expansion. This study provides a framework for applying high-throughput DNA sequencing technologies for future investigations of ticks, which are common vectors of diseases.