The study generated a phenotypic dataset for 550 Lactuca accessions, including 20 wild relatives, and applied an iterative two‑step GWAS using a jointly processed SNP set for cultivated lettuce (L. sativa) and its wild progenitor (L. serriola) to dissect trait loci. Known and novel QTLs for anthocyanin accumulation, leaf morphology, and pathogen resistance were identified, with several L. serriola‑specific QTLs revealing unique genetic architectures, underscoring the breeding value of wild lettuce species.