AbstractGrapevine is one of the most economically important crops worldwide. However, the previous versions of the grapevine reference genome tipically consist of thousands of fragments with missing centromeres and telomeres, limiting the accessibility of the repetitive sequences, the centromeric and telomeric regions, and the study of inheritance of important agronomic traits in these regions. Here, we assembled a telomere-to-telomere (T2T) gap-free reference genome for the cultivar PN40024 using PacBio HiFi long reads. The T2T reference genome (PN_T2T) is 69 Mb longer with 9018 more genes identified than the 12X.v0 version. We annotated 67% repetitive sequences, 19 centromeres and 36 telomeres, and incorporated gene annotations of previous versions into the PN_T2T assembly. We detected a total of 377 gene clusters, which showed associations with complex traits, such as aroma and disease resistance. Even though PN40024 derives from nine generations of selfing, we still found nine genomic hotspots of heterozygous sites associated with biological processes, such as the oxidation–reduction process and protein phosphorylation. The fully annotated complete reference genome therefore constitutes an important resource for grapevine genetic studies and breeding programs.