BackgroundThe first telomere-to-telomere (T2T) human genome assembly (T2T-CHM13) release is a milestone in human genomics. The T2T-CHM13 genome assembly extends our understanding of telomeres, centromeres, segmental duplication, and other complex regions. The current human genome reference (GRCh38) has been widely used in various human genomic studies. However, the large-scale genomic differences between these two important genome assemblies are not characterized in detail yet.ResultsHere, in addition to the previously reported “non-syntenic” regions, we find 67 additional large-scale discrepant regions and precisely categorize them into four structural types with a newly developed website tool called SynPlotter. The discrepant regions (~ 21.6 Mbp) excluding telomeric and centromeric regions are highly structurally polymorphic in humans, where the deletions or duplications are likely associated with various human diseases, such as immune and neurodevelopmental disorders. The analyses of a newly identified discrepant region—theKLRCgene cluster—show that the depletion ofKLRC2by a single-deletion event is associated with natural killer cell differentiation in ~ 20% of humans. Meanwhile, the rapid amino acid replacements observed withinKLRC3are probably a result of natural selection in primate evolution.ConclusionOur study provides a foundation for understanding the large-scale structural genomic differences between the two crucial human reference genomes, and is thereby important for future human genomics studies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-023-02995-w.