The article reports the assembly and annotation of a high-quality reference genome for maize, a genetically and agriculturally important model species, using single-molecule real-time sequencing and high-resolution optical mapping. The new assembly, named B73 RefGen_v4, features a 52-fold increase in contig length compared to the previous reference genome, with improved resolution of intergenic spaces and centromeres. The study identified over 130,000 intact transposable elements and updated gene annotations using 11,000 full-length transcripts. Comparative optical mapping of two additional maize lines revealed a prevalence of deletions in regions of low gene density and lineage-specific genes. The improved genome assembly will enhance the understanding of genetic diversity and phenotypic variation in maize and other economically important plants.The article reports the assembly and annotation of a high-quality reference genome for maize, a genetically and agriculturally important model species, using single-molecule real-time sequencing and high-resolution optical mapping. The new assembly, named B73 RefGen_v4, features a 52-fold increase in contig length compared to the previous reference genome, with improved resolution of intergenic spaces and centromeres. The study identified over 130,000 intact transposable elements and updated gene annotations using 11,000 full-length transcripts. Comparative optical mapping of two additional maize lines revealed a prevalence of deletions in regions of low gene density and lineage-specific genes. The improved genome assembly will enhance the understanding of genetic diversity and phenotypic variation in maize and other economically important plants.