Exposure of Arabidopsis thaliana (L.) photorespiration mutants to air leads to a rapid decline in the Fv/Fm chlorophyll fluorescence parameter, reflecting a decline in PSII function and an onset of photoinhibition. This paper demonstrates that chlorophyll fluorescence imaging of Fv/Fm can be used as an easy and efficient means of detecting Arabidopsis mutants that are impaired in various aspects of photorespiration. This screen was developed to be sensitive and high throughput by the use of exposure to zero CO2 conditions and the use of array grids of 1-week-old Arabidopsis seedlings as the starting material for imaging. Using this procedure, we screened ~25 000 chemically mutagenised M2 Arabidopsis seeds and recovered photorespiration phenotypes (reduction in Fv/Fm at low CO2) at a frequency of ~4 per 1000 seeds. In addition, we also recovered mutants that showed reduced Fv/Fm at high CO2. Of this group, we detected a novel ‘reverse photorespiration’ phenotype that showed a high CO2 dependent reduction in Fv/Fm. This chlorophyll fluorescence screening technique promises to reveal novel mutants associated with photorespiration and photoinhibition.