Lettuce (Lactuca sativa L.) is one of the most important leafy vegetables in the world due to its health related benefits. Bitter sesquiterpene lactones (BSLs) are crucial secondary metabolites of lettuce which are useful in many physiological activities, and also has been used to relieve pain. This study was conducted to appraise the variability of phenotypic characteristics and the total content of BSLs in fifty lettuce germplasm. The germplasm collections exhibited wide variation in both qualitative and quantitative characters. The average plant weight was 333.3 g with a range from 60.0 to 700.0 g and the lactucin content was varied from 3.4 (IT265031) to 448.6 (IT300287) μg/g DW with an average concentration of 94.0 μg/g DW. Whereas lactucopicrin ranged from 6.8 (IT217859) to 2,714.9 (Superseonpung) μg/g DW with an average concentration of 805.8 μg/g DW. Total BSLs content was ranged from 19.6 (IT217859) to 2,821.9 (Superseonpung) μg/g DW with the average concentration of 899.8 μg/g DW. Significant (p≤0.05) differences in lactucin, lactucopicrin and total BSLs content were found among the germplasm collections. Leafy type lettuce germplasm collections originated from South Korea revealed the highest average total BSLs content (2,821.9 μg/g DW). Principal component analyses of lactucin and lactucopicrin generated based on leaf color and plant growth type yielded two principal components, PC1 and PC2, which accounted for 60.8 and 39.2 % of the total variance, respectively. Our study provides fundamental insights on phenotypic characteristics and the total content of lactucin and lactucopicrin of lettuce germplasm which may further help researchers and breeders to produce improved lettuce varieties.