Abstract Topologically associating domains (TADs) were recently identified as fundamental units of three-dimensional eukaryotic genomic organization, though our knowledge of the influence of TADs on genome evolution remains preliminary. To study the molecular evolution of TADs in Drosophila species, we constructed a new reference-grade genome assembly and accompanying high-resolution TAD map for D. pseudoobscura . Comparison of D. pseudoobscura and D. melanogaster , which are separated by ∼49 million years of divergence, showed that ∼30-40% of their genomes retain conserved TADs. Comparative genomic analysis of 17 Drosophila species revealed that chromosomal rearrangement breakpoints are enriched at TAD boundaries but depleted within TADs. Additionally, genes within conserved TADs exhibit lower expression divergence than those located in nonconserved TADs. Furthermore, we found that a substantial proportion of long genes (&gt;50 kbp) in D. melanogaster (42%) and D. pseudoobscura (26%) constitute their own TADs, implying transcript structure may be one of the deterministic factors for TAD formation. Using structural variants (SVs) identified from 14 D. melanogaster strains, its 3 closest sibling species from the D. simulans species complex, and two obscura clade species, we uncovered evidence of selection acting on SVs at TAD boundaries, but with the nature of selection differing between SV types. Deletions are depleted at TAD boundaries in both divergent and polymorphic SVs, suggesting purifying selection, whereas divergent tandem duplications are enriched at TAD boundaries relative to polymorphism, suggesting they are adaptive. Our findings highlight how important TADs are in shaping the acquisition and retention of structural mutations that fundamentally alter genome organization.

Topologically associating domains and their role in the evolution of genome structure and function in Drosophila