The highly transformable maize inbred line LH244 represents an attractive model for gene discovery and genome engineering. However, the lack of a high‐quality genome assembly has limited its utility in functional genomics research. Here, we present a 2.29 Gb near‐complete assembly of the LH244 maize genome, with an overall base accuracy of 99.998%. Except for five gaps associated with super‐long thymine–adenine–guanine (TAG) repeat arrays, all the genome sequences were assembled from telomere to telomere (T2T). Comparative analysis revealed high genetic similarity between LH244 and B73, including 80.06% genome‐wide synteny and 90.92% of genes nearly identical. The LH244 genome was also compared with the complete Mo17 genome and revealed extensive intraspecific genomic variations. A total of 14 megabase‐scale structural variations (SVs) were identified, including a 3.15 Mb insertion, harboring 95 genes, within the 45S rDNA array of LH244 but not in the Mo17 genome. In addition, there were five knob arrays, with an average size of 21.76 Mb and the longest of 38.70 Mb, only existing in the LH244 genome. Despite the substantial variation in knob abundance, knob‐6S and knob‐8L were highly conserved between LH244 and Mo17, showing strong synteny and sequence identity, as well as consistent insertion patterns of genes and transposable elements (TEs). Overall, our study provides a near‐complete reference genome of an important transformable maize germplasm, which will serve as a much‐needed resource for functional genomics and genome editing of maize.

The gene-editing tool Cas-SF01 was optimized to maximize its efficiency in maize. The Cas-SF01-TREX2 configuration was superior in enabling high-purity gene mutations. This toolkit enabled commercial maize to flower seven days earlier without yield loss, thereby securing harvests and accelerating crop breeding.