Lettuce is one of the economically important leaf vegetables and is cultivated mainly in temperate climate areas. Cultivar identification based on the distinctness, uniformity, and stability (DUS) test is a prerequisite for new cultivar registration. However, DUS testing based on morphological features is time-consuming, labor-intensive, and costly, and can also be influenced by environmental factors. Thus, molecular markers have also been used for the identification of genetic diversity as an effective, accurate, and stable method. Currently, genome-wide single nucleotide polymorphisms (SNPs) using next-generation sequencing technology are commonly applied in genetic research on diverse plant species. This study aimed to establish an effective and high-throughput cultivar identification system for lettuce using core sets of SNP markers developed by genotyping by sequencing (GBS). GBS identified 17 877 high-quality SNPs for 90 commercial lettuce cultivars. Genetic differentiation analyses based on the selected SNPs classified the lettuce cultivars into three main groups. Core sets of 192, 96, 48, and 24 markers were further selected and validated using the Fluidigm platform. Phylogenetic analyses based on all core sets of SNPs successfully discriminated individual cultivars that have been currently recognized. These core sets of SNP markers will support the construction of a DNA database of lettuce that can be useful for cultivar identification and purity testing, as well as DUS testing in the plant variety protection system. Additionally, this work will facilitate genetic research to improve breeding in lettuce.