Abstract: In present studies, level of toxicity of hygromycin was optimized for local inbred line of maize (Zea mays L.). The immature embryos were isolated and cultured on N6 media modified with 1.0 mg L-1 2,4-D, 25 mM L-Proline and 100 mg L-1 Casein Hydrolysate and containing different concentrations of hygromycin. These immature embryos were observed for callus formation on working concentrations of 0, 20, 40, 60, 80, 100 and 120 mg L-1 hygromycin. The results showed that 97.3 38.5 and 32.5% calli survived on media containing 0, 20 and 40 mg L-1 hygromycin. Hygromycin concentration of 60 mg L-1 and above was effective for BR-6 as no callus formation was observed at this concentration or the immature embryos were completely dead. It was also observed that although 32.8% calli survived on media containing 40 mg L-1 hygromycin, these calli were slower in growth, smaller in size and pale in color as compared to calli containing (hph) gene on same media. On the basis of these results 40 mg L-1 hygromycin seems suitable for identification of putative transformants.