Seed & Variety Service for Plant Variety Protection (PVP). We previously constructed DNA profile database for identifying 159 lettuce varieties using 60 simple sequence repeat (SSR) markers. In this study, we selected optimum markers from previously applied markers and new SSR markers for the standardization of DNA profile database of 65 commercial lettuce varieties containing 18 PVP varieties distributed in Korea. Twenty-eight SSR markers from 60 SSR markers were selected for characterization with 65 commercial lettuce varieties according to the reproducibility, polymorphism, band pattern of marker and easiness of scoring. Out of 156 SSR primers, we additionally selected 11 new SSR primer pairs showed polymorphisms between 8 varieties and repetitive reproducibility on capillary electrophoresis system. Totally 127 polymorphic amplified fragments were obtained using 39 SSR markers. Two to seven SSR alleles were detected for each locus with an average of 3.3 alleles per locus. Average polymorphism information content was 0.517, ranging from 0.281 to 0.771. Genetic distance of clusters ranged from 0.29 to 0.96 by unweighted pair-group method with arithmetical average based on Jaccard’s distance coefficients. A total of 65 commercial lettuce varieties were discriminated by 39 SSR marker genotypes. These SSR profile database developed will be continually characterized for the standardization of DNA profiles for lettuce commercial varieties