Stomata regulates conductance, transpiration and photosynthetic traits in plants. Increased stomatal density may contribute to enhanced water loss and thereby help improve the transpirational cooling process and mitigate the high temperature-induced yield losses. However, genetic manipulation of stomatal traits through conventional breeding still remains a challenge due to problems involved in phenotyping and the lack of suitable genetic materials. Recent advances in functional genomics in rice identified major effect genes determining stomatal traits, including its number and size. Widespread applications of CRISPR/Cas9 in creating targeted mutations paved the way for fine tuning the stomatal traits for enhancing climate resilience in crops. In the current study, attempts were made to create novel alleles ofOsEPF1(Epidermal Patterning Factor), a negative regulator of stomatal frequency/density in a popular rice variety, ASD 16, using the CRISPR/Cas9 approach. Evaluation of 17 T0progenies identified varying mutations (seven multiallelic, seven biallelic and three monoallelic mutations). T0mutant lines showed a 3.7–44.3% increase in the stomatal density, and all the mutations were successfully inherited into the T1generation. Evaluation of T1progenies through sequencing identified three homozygous mutants for one bp insertion. Overall, T1plants showed 54–95% increased stomatal density. The homozygous T1lines (# E1-1-4, # E1-1-9 and # E1-1-11) showed significant increase in the stomatal conductance (60–65%), photosynthetic rate (14–31%) and the transpiration rate (58–62%) compared to the nontransgenic ASD 16. Results demonstrated that the genetic alterations inOsEPF1altered the stomatal density, stomatal conductance and photosynthetic efficiency in rice. Further experiments are needed to associate this technology with canopy cooling and high temperature tolerance.