A new, highly improved reference genome for maize (Zea mays) has been developed using single-molecule real-time (SMRT) sequencing and high-resolution optical mapping. This assembly significantly enhances the contiguity and accuracy of the genome, with a 52-fold increase in contig length compared to the previous reference genome. The new assembly includes 2,958 contigs, with half exceeding 1.2 Mb, and is integrated with a high-quality optical map to create 625 scaffolds. The final assembly consists of 2,522 gaps, with 1,115 of these estimated using optical genome maps. The new reference genome has 240-fold higher contiguity than the previously published short-read assembly of maize cultivar PH207. The improved assembly allows for more accurate gene annotations, with 111,000 full-length transcripts used to update gene models. This results in a more complete annotation of alternative splicing and regulatory regions, with over 70% of genes supported by full-length transcripts. The assembly also reveals more than 130,000 intact transposable elements, providing insights into maize lineage-specific expansions. The new reference genome has been used to analyze structural variations in two additional maize inbred lines, Ki11 and W22, revealing a high level of structural diversity. The assembly also provides a more accurate representation of centromeres and telomeres, with the ends of chromosomes properly identified in 14 out of 20 chromosome arms. The improved genome assembly enhances our understanding of maize genetics and genomics, enabling better identification and prediction of functional genetic variation. It also provides a more accurate framework for studying the evolution of maize and its relationship to other grass species. The new reference genome is expected to improve agricultural research and crop improvement efforts by providing a more complete and accurate genetic resource for maize.A new, highly improved reference genome for maize (Zea mays) has been developed using single-molecule real-time (SMRT) sequencing and high-resolution optical mapping. This assembly significantly enhances the contiguity and accuracy of the genome, with a 52-fold increase in contig length compared to the previous reference genome. The new assembly includes 2,958 contigs, with half exceeding 1.2 Mb, and is integrated with a high-quality optical map to create 625 scaffolds. The final assembly consists of 2,522 gaps, with 1,115 of these estimated using optical genome maps. The new reference genome has 240-fold higher contiguity than the previously published short-read assembly of maize cultivar PH207. The improved assembly allows for more accurate gene annotations, with 111,000 full-length transcripts used to update gene models. This results in a more complete annotation of alternative splicing and regulatory regions, with over 70% of genes supported by full-length transcripts. The assembly also reveals more than 130,000 intact transposable elements, providing insights into maize lineage-specific expansions. The new reference genome has been used to analyze structural variations in two additional maize inbred lines, Ki11 and W22, revealing a high level of structural diversity. The assembly also provides a more accurate representation of centromeres and telomeres, with the ends of chromosomes properly identified in 14 out of 20 chromosome arms. The improved genome assembly enhances our understanding of maize genetics and genomics, enabling better identification and prediction of functional genetic variation. It also provides a more accurate framework for studying the evolution of maize and its relationship to other grass species. The new reference genome is expected to improve agricultural research and crop improvement efforts by providing a more complete and accurate genetic resource for maize.