Citrus species are economically and nutritionally vital, with their fruits cultivated globally. Despite the publication of multiple genomes for Citrus, high-quality assemblies that achieve both haplotype resolution and telomere-to-telomere (HR-T2T) continuity remain scarce—pummelo (Citrus maxima) being a notable example. Compounded by limitations in gene annotation quality, these gaps hinder functional genomic research and genomics-assisted breeding. Here, we report the first high-quality HR–T2T genome assembly of pummelo, generated using PacBio HiFi and Oxford Nanopore sequencing. The two haplotype assemblies presented contig N50 values of 38.58 and 32.57 Mb, completeness scores of 99.36% and 99.66%, and nucleotide accuracies of 99.99994% and 99.99997%, respectively. We developed HapGene, a haplotype-aware annotation pipeline that integrates short-read RNA-Seq and long-read Iso-Seq data to enable unbiased annotation. Benchmarking showed HapGene captured 3% to 10% of genes missed or misannotated by conventional pipelines and reduces false haplotype-specific genes by 4- to 5-fold. Leveraging 380 Gb of newly sequenced and 2792 Gb of public transcriptomic data, we comprehensively annotated protein-coding and non-coding genes across three major Citrus crops (sweet orange, pummelo, and mandarin). This effort revealed 18 757–21 083 alternative splicing events, 1725–1853 resistance gene analogues, and 2392–3757 long intergenic RNAs (lincRNAs). Genomic and transcriptomic characterization of lincRNAs indicated their functional innovation (many associated with stress responses) in Citrus. Additionally, we revealed around one-third of genes exhibited tissue-specific allelic differential expression. Our work provides a critical genomic resource and analytical tool to advance Citrus genomic research, thereby driving progress in functional and evolutionary genomics while laying a robust foundation for precise genomics-assisted breeding.

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