Polyploidy induction could increase shallot bulb-size to raise consumer preference and local shallot productivity. The research aimed to obtain an effective method of polyploidy induction on callus of onion (Allium cepa) var. Bima Brebes. The experiment was consisted of two experimental steps, which were callus induction of onion and polyploid induction of the callus. A 1×1 cm callus was treated by two drops of oryzalin with concentrations 0, 25, 50, 75, 100, and 120 μM. The ploidy level was identified based on morphological trait, stomatal analysis and DNA content using a flow cytometry. The results showed callus diameter, number of green spots, and number of shoots were decreased with increasing oryzalin concentration. The planlet leaves regenerated from oryzalin treated callus were darker than that of control. The flow cytometry analysis showed that planlets with 75 μM oryzalin was tetraploid, had longer and wider stomata than that of the control.