Two major QTL, one for shelf life that corresponds to qSL4 and one, qDEV7, for developmental rate, were identified. Associated markers will be useful in breeding for improved fresh-cut lettuce. Fresh-cut lettuce in packaged salad can have short shelf life, and visible deterioration may start within a week after processing. Yield and developmental rate are an important aspect of lettuce production. Genetic diversity and genome-wide association studies (GWAS) were performed on 493 accessions with the genotypic data of 4615 high-quality single nucleotide polymorphism markers. Population structure (Q), principal component (PC), and phylogenetic analyses displayed genetic relationships associated with lettuce types and geographic distribution. Data for shelf life, yield, developmental rate, and their stability indices were used for statistical analysis, and GWAS was performed by general and mixed linear models. The genetic relationship among the individuals was incorporated into the models using kinship matrix, PC, and Q. Broad-sense heritability (H2) across environments was 0.43 for shelf life, 0.36 for yield, and 0.60 for developmental rate. There was a negative correlation between yield and developmental rate. Significant marker-trait association (SMTA) was detected for shelf life on chromosome 4. The most significant quantitative trait locus (QTL, qSL4, P = 2.23E-17) explained 24% of the total phenotypic variation (R2). The major QTL for developmental rate was detected on chromosome 7 (qDEV7, P = 2.43E-16, R2 = 17%), while additional QTLs with smaller effect were found in all chromosomes. No SMTA was detected for yield. The study identified lettuce accessions with extended and stable shelf life, stable yield, and desirable developmental rate. Molecular markers closely linked to traits can be applied for selection of preferable genotypes and for identification of genes associated with these traits.