An Australian chickpea pan-genome provides insights into genome organization and offers opportunities for enhancing drought adaptation for crop improvement.

Chickpea (Cicer arietinum L.) is an important legume crop that has been subjected to intensive breeding, resulting in limited genetic diversity. Australia is the world's second largest producer and the leading exporter of chickpea; the genomic architecture of its cultivars remains largely unexplored. This knowledge gap hinders efforts to enhance their genetic potential for production, protection, and stress adaptation. To address this, we generated high-quality genome assemblies and annotations for 15 leading Australian chickpea cultivars using single-tube long-fragment read technology. The pan-genome analysis identified 34 345 gene families, including 13 986 dispensable families enriched for genes associated with key agronomic traits. Comparative genomic analysis revealed ~2.5 million single-nucleotide polymorphisms, nearly 200 000 insertions/deletions, and over 280 000 structural variations. These variations were found in key flowering time genes, seed weight-related genes, and disease resistance genes, providing insights into the genetic diversity underlying these critical traits. Haplotype analysis of key genes within the 'QTL-hotspot' region revealed the absence of superior haplotypes in Australian cultivars. Validation using Kompetitive allele-specific PCR markers confirmed these findings, highlighting the need to introduce beneficial haplotypes from diverse accessions to enhance drought tolerance in Australian chickpea cultivars. The genomic resources generated in this study provide valuable insights into chickpea genetic diversity and offer potential avenues for crop improvement.