High temperature stress at flowering can adversely affect rice yield, largely due to failure of fertilization. Oxidative damage can be a major reason inducing spikelet sterility in rice. In the present study, the effect of high temperatures on antioxidant metabolism in rice spikelets was characterised using nine different genotypes. Exposure to different temperatures at flowering stage revealed significant differences among various antioxidant enzymes in spikelets, both quantitatively and qualitatively. Spikelets of susceptible genotypes withstood temperature stress of up to 35 °C, those of moderately tolerant between 35 °C and 38 °C and those of tolerant genotypes up to 40 °C. Presence or absence, and changes in the isozyme intensities were consistent with alterations in their activities. Superoxide dismutase (SOD) isozymes II and III were present after exposure at 30 °C and 35 °C, while SOD I appeared above 40°C. Intensities of catalase isozymes I and III and the only isozyme of ascorbate peroxidase altered, while the only isozyme of guaical peroxidase and two (III and IV) of the four isozymes of catechol peroxidase disappeared after high temperature exposure of 45 °C. Thus, this work provides an evidence of the role of antioxidant metabolism in spikelets under high temperature stress conditions. Hence, changes in antioxidant isozymes in rice spikelets can be used as a biomarker for characterizing high temperature stress tolerance in rice spikelets.