Characterization of genetic differences between lineages of the dimorphic human-pathogenic fungus Paracoccidioides can identify changes linked to important phenotypes and guide the development of new diagnostics and treatments. In this article, we compared genomes of 31 diverse isolates representing the major lineages of Paracoccidioides spp. and completed the first annotated genome sequences for the PS3 and PS4 lineages. We analyzed the population structure and characterized the genetic diversity among the lineages of Paracoccidioides , including a deep split of S1 into two lineages (S1a and S1b), and differentiated S1b, associated with most clinical cases, as the more highly recombining and diverse lineage. In addition, we found patterns of positive selection in surface proteins and secreted enzymes among the lineages, suggesting diversifying mechanisms of pathogenicity and adaptation across this species complex. These genetic differences suggest associations with the geographic range, pathogenicity, and ecological niches of Paracoccidioides lineages.