Spot blotch caused by Bipolaris sorokiniana has spread to more than 9 million ha of wheat in the warm, humid areas of the Eastern Gangetic Plains (EGP) of South Asia and is a disease of major concern in other similar wheat growing regions worldwide. Differential lignin content in resistant and susceptible genotypes and its association with free radicals such as hydrogen peroxide (H2O2), superoxide (O2 -) and hydroxyl radical (OH-) were studied after inoculation under field conditions for two consecutive years. H2O2 significantly influenced lignin content in flag leaves, whereas there was a negative correlation among lignin and H2O2 to the Area Under Disease Progress Curve (AUDPC). The production of H2O2 was higher in the resistant genotypes than susceptible ones. The O2 - and OH- positively correlated with AUDPC but negatively with lignin content. This study illustrates that H2O2 has a vital role in prompting lignification and thereby resistance to spot blotch in wheat. We used cluster analysis to separate the resistant and susceptible genotypes by phenotypic and biochemical traits. H2O2 associated lignin production significantly reduced the number of appressoria and penetration pegs. We visualized the effect of lignin in disease resistance using differential histochemical staining of tissue from resistant and susceptible genotypes, which shows the variable accumulation of hydrogen peroxide and lignin around penetration sites.