SummaryOncidium is one of the most popular ornamental orchids. However, it is difficult to differentiate one cultivar from another based only on phenotypic traits. As Plant Breeders’ Rights evolve, in particular since the advent of essential derivations, the importance of early differentiation of crop cultivars increases. In this report,AFLP markers were used, with 21 primer pair combinations, to fingerprint two cultivars from Oncidium Gower Ramsey hybrids and two cultivars from O. Sweet Sugar hybrids with the restriction endonucleases Eco RI and Mse I. A total of 1,091 AFLP bands were obtained. More polymorphic bands (209) were detected between the two hybrids than between the two cultivars of O. Gower Ramsey (25 bands), or between the two cultivers of O. Sweet Sugar (18). The most significant polymorphic AFLP bands (11) were cloned and sequenced for conversion into 12 sequence-tagged site (STS) markers. These STS markers were then applied to the identification of 49 cultivars from four Oncidium hybrids including O. Gower Ramsey, O. Sweet Sugar, O. Kaizumic Delight and O. Sharry Baby. Three STS markers (P03A, P03B and P10) were successfully identified to differentiate the three hybrids (O. Gower Ramsey, O. Sweet Sugar, and O. Sharry Baby) and three cultivars of O. Sweet Sugar (‘Angel’, ‘Emperor’ and ‘Million Coin’). In conclusion, STS molecular markers, developed through AFLP fingerprinting, could prove useful to distinguish new cultivars of Oncidium orchid.