Polyphenol oxidase partial gene PG-PPO was cloned and characterized from Pennisetum glaucum (pearl millet) which showed 42% identity to a PPO sequence isolated from wheat at the region of Copper B with a score of 40 and e-value of 2.8. Multiple sequence alignment results revealed similarity to polyphenol oxidase (PPO) sequences from wheat, trifolium, lettuce, apricot, tobacco, tomato, pokeweed, apple, grape and poplar especially at the Copper B region of PPO. The 395 bp pearl millet PPO sequence was AT rich (53.3%) and contained the highly conserved amino acids of histidine-rich copper binding sites similar to PPO sequences from other crops. Results also indicated that PPO in pearl millet exists in multi copy. The role of the isolated PPO gene during pearl millet-downy mildew interaction was analyzed and the results showed significantly higher and rapid accumulation of PPO mRNAs in resistant pearl millet seedlings inoculated with Sclerospora graminicola in comparison to the susceptible control, demonstrating that the PPO plays a prominent role in pearl millet defense against pathogens, particularly downy mildew pathogen.