The blacklegged tick, Ixodes scapularis, is a major disease vector in the United States with the vectorial capacity to transmit seven human pathogens, including those causing anaplasmosis, babesiosis, Borrelia miyamotoi disease, Powassan virus disease, ehrlichiosis, and Lyme disease. Lyme disease is a slow-moving epidemic in North America with an estimated 400,000 annual infections mostly caused by I. scapularis and over 90% of infections occurring in the northeastern and midwestern U.S. The range of endemic Lyme disease is expanding from historical regions as northern I. scapularis populations are being established in new counties and are potentially hybridizing with southern I. scapularis populations. Northern and southern populations of I. scapularis exhibit significant differences in host-seeking behaviors, which appear to correlate with higher vectorial capacity for Lyme disease pathogen Borrelia burgdorferi sensu strictro among northern ticks. These behavioral differences are partially caused by genetic drivers which have not yet been identified. In addition to a limited understanding of the extent of population genetic differentiation throughout the genome of I. scapularis, there is also a lack of research done on the genetic differentiation of I. scapularis males and females. In this dissertation, we have identified genomic regions within the I. scapularis genome that are highly variable among northern and southern populations. These regions are identified as targets for future studies to link host-seeking behaviors to genotypes. We have also quantified the amount of whole genome variation occurring within and among populations using multiple sequencing techniques and analysis pipelines, establishing that the population differentiation between northern and southern populations of I. scapularis is highly robust and detectable via multiple methods. Finally, we have identified sex-specific regions of the I. scapularis genome including a chromosome-sized scaffold that is the X-specific region of the X chromosome, and male-specific alleles that we used to develop a sex differentiating PCR assay for non-sexually dimorphic life stages of I. scapularis. This male-specific PCR assay can be implemented to investigate differences in microbiome, behavior, host association, and other important components of vectorial capacity that may differ between the sexes at early life stages, which have been inaccessible to research until now.