The Lactuca genus, belonging to one of the largest plant families, Asteraceae, encompasses over 97 species with considerable economic importance. Several species of the genus Lactuca, such as L. sativa, have been cultivated since ancient times and are globally significant as leafy vegetables. The nutritional value and taste of these species are paramount considerations for both breeders and consumers. The perceived bitter taste, primarily imparted by varying quantities of sesquiterpene lactones, markedly impacts consumption patterns. Bitter sesquiterpene lactones, including lactucin, 8-deoxylactucin, and lactucopicrin in lettuce, play pivotal roles in plant defense mechanisms against herbivores and pathogens. To elucidate the genetic basis of differences in the contents of bitter sesquiterpene lactones, we conducted a comprehensive genome-wide association study using genotyping-by-sequencing on a diverse panel of 1,730 Lactuca germplasm collections, including six species (L. sativa, L. serriola, L. saligna, L. indica, L. altaica, and L. livida). A total of 7,321 high-quality SNPs were identified and used to perform association analysis for bitter sesquiterpene lactone levels via a mixed linear model. Candidate genes for 11 associated loci were identified through detailed annotation. These findings will be useful for breeding strategies aimed at cultivating lettuce with more favorable taste profiles. These findings herald a new era in the cultivation of lettuce varieties, specifically engineered to enhance sleep quality, marking a significant stride towards leveraging natural ingredients for improved sleep patterns in consumers.