Lettuce (Lactuca sativa) is a globally cultivated leafy vegetable with leafy morphology critically influencing consumer preference and market value. Despite the agronomic importance of leaf traits, the genetic basis underlying their diversity remains poorly characterized. To address this, we resequenced 811 accessions collected from major lettuce production areas as well as the relative wild species, and developed a publicly accessible core collection of 268 accessions that captures 99.4% of the total genetic variation. Phenotypic evaluation of 16 leaf morphological traits across two growing seasons identified significant correlations, including negative associations between plant width and anthocyanin content, and positive correlations between apical margin incision and multiple traits. Population structure analysis revealed frequent introgression events from looseleaf type into domesticated varieties (butterhead, crisphead, romaine, and stem lettuce), highlighting dynamic gene flow during breeding. Genome-wide association studies (GWAS) pinpointed 13 robust quantitative trait loci (QTLs) and candidate genes regulating leaf morphology, including a validated anthocyanin biosynthesis regulator (ANS). Notably, we pinpointed the causal gene genotypes responsible for leaf anthocyanin coloration. Leveraging these findings, we successfully aggregated favorable alleles through genomic design breeding to develop a novel high-anthocyanin variety binfen5 with desirable leaf morphology. This integrative approach demonstrates the value of core germplasms and genomic tools for accelerating lettuce improvement.

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