Genetic diversity, population structure, and genome-wide marker-trait association analyses were conducted on a special collection of 298 homozygous lettuce(Lactuca sativa L.) lines. Each of these lines was derived from a single plant that had been genotyped with 384 SNP markers using LSGermOPA. They included 122 butterhead, 53 romaine, 63 crisphead, 53 leaf and 7 stem types. Genetic diversity among these plants was assessed by pairwise comparison based on 322 high-quality SNP markers selected from 384 SNPs. Only 258 unique genotypes were identified among the 298 lines because 26 pairs or small groups(a total of 66 lines) shared identical genotypes. The average genetic similarity coefficient(GS) among these unique genotypes was 63.9% with a range of 40.6% to 99.8%. A phylogenetic tree was constructed based on the genotypic data. The most likely number of populations was estimated to be two or six. Association analysis between the 322 SNP markers and 10 phenotypic traits using the 258 homozygous lines was performed by three different methods: single factor analysis, general linear model analysis, and mixed linear model analysis. Nine significant marker-trait associations(SMTAs) were detected at P < 0.0001 with all three methods and also when considering kinship and/or population structure for this collection, with five SMTAs for seed coat color, one for leaf undulation, two for leaf anthocyanin, and one for stem anthocyanin. These markers will be useful in marker-assisted selection after further validation with segregating populations. Genetic diversity, population structure, and genome-wide marker-trait association analyzes were conducted on a special collection of 298 homozygous lettuce (Lactuca sativa L.) lines. Each of these lines was derived from a single plant that had been genotyped with 384 SNPs They included 122 butterhead, 53 romaine, 63 crisphead, 53 leaf and 7 stem types. Genetic diversity among these plants was assessed by pairwise comparison based on 322 high-quality SNP markers selected from 384 SNPs. Only 258 unique genotypes were identified among the 298 lines because 26 pairs or small groups (a total of 66 lines) shared identical genotypes. The average genetic similarity coefficient (GS) among these unique genotypes was 63.9% with a range of 40.6% to 99.8%. A phylogenetic tree was constructed based on the genotypic data. The most likely number of populations was estimated to be two or six. Association analysis between the 322 SNP markers and 10 phenotypic traits using the 258 ho Nine significant marker-trait associations (SMTAs) were detected at P <0.0001 with all three methods and also when considering kinship and / or population structure for this collection, with five SMTAs for seed coat color, one for leaf undulation, two for leaf anthocyanin, and one for stem anthocyanin. These markers will be useful in marker-assisted selection after further validation with segregating populations.