Lettuce (Lactuca sativa L.) is one of the most important leafy vegetable crops worldwide. Soil salinity adversely affects lettuce production leading to considerable yield losses. Identification of genetic loci controlling salt tolerance will facilitate molecular marker development and thereby assist breeders in developing lettuce cultivars with salt tolerance. Accordingly, we conducted a genome-wide association study (GWAS) to identify marker-trait association for salt tolerance at the seedling stage using 409 diverse lettuce accessions and 56,820 high-quality single nucleotide polymorphism (SNP) markers obtained through genotype-by-sequencing technology. Several statistical models, including GLM, MLM, FarmCPU, and BLINK were employed using the GAPIT version 3 software tool for GWAS. Based on three important seedling stage traits affected by salinity, i.e., shoot fresh weight (FW), shoot dry weight (DW) and chlorophyl index (SPAD), 13 significant salt tolerance related SNPs representing 10 QTLs were identified on lettuce chromosomes 1, 3, 4, 6, 7, 8 and 9. Notably, a major QTL on chromosome 4, encompassing four significant SNPs within a 116 bp region of the lettuce reference genome (v8), explained 49% of the phenotypic variation for FW. The identified salt tolerance-related QTLs provide a valuable resource for developing assays for marker-assisted selection to breed lettuce cultivars with improved salt tolerance.