Amplified fragment length polymorphism (AFLP) was used to assess genetic relatedness among 23 popular commercial cultivars of lychee (Litchi chinensis) and identify the markers associated with small-seeded cultivars. A combination of three primer pairs with high discriminatory ability was selected for resolving the identity of lychee cultivars. The marker system resolved the instances of synonymies and homonymies. The two cultivars, Dehrarose and Dehradun, whose names are often used interchangeably, could be discriminated by AFLP. A cultivar, Calcuttia (H), grown in the Punjab region was found genetically very similar to China (M) of Bihar region in India, suggesting that both could be the same clone. A number of cultivars with contrasting seed size were included in the study. The cultivars Bedana, Seedless Late, and China were positioned in one group, unlike the grouping previously reported based on morphological traits. The AFLP fingerprinting revealed five marker fragments exclusive to the genotypes, which bear fruit with small seed and large pulp.