Abstract Drosophila pseudoobscura is a long-standing model organism in evolutionary genetics because natural populations segregate for an inversion polymorphism on the third chromosome. In addition, D. pseudoobscura has a neo-X chromosome that was formed by an X-autosome fusion, which segregates for a sex-ratio drive allele. Previous genome-wide studies of DNA sequence variation in D. pseudoobscura have focused on individual chromosomes or did not use chromosome-scale reference genomes. To address these shortcomings, we generated a new D. pseudoobscura population genetic resource by sequencing the genomes of over 60 inbred lines sampled across the species’ geographic range in North America. Using these data, we examined patterns of nucleotide diversity and population structure across the entire genome. Tajima’s D was negative across most of the genome, consistent with a recent population expansion. However, there was substantial heterogeneity of D across chromosomes, suggesting distinct evolutionary dynamics across the genome. We found no strong evidence of population structure across most chromosomes, consistent with a near panmictic population. In contrast, we identified population structure on the third chromosome, which we attributed to the inversion polymorphism and used to infer the arrangements carried by the strains we sampled. Our analysis therefore demonstrates that tests for population structure can identify polymorphic chromosomal rearrangements. The population genomic data we have collected is publicly available and will support future research on genome evolution, local adaptation, and sex chromosome evolution in D. pseudoobscura .