The diallel mating design is an important tool used by plant breeding programs to obtain information on trait inheritance. Knowledge of gene action, heritability and genetic advance from selection is a prerequisite for starting a breeding program for developing varieties of maize. Five maize S₇ lines and their F₂ progenies were studied in a 5 × 5 half-diallel crossing design to evaluate the gene action and the heritability of biochemical and physiological traits. Parents and their F₂ hybrids were planted in a randomized complete block design with three replications at the Research Farm of Graduate University of Advanced Technology (Kerman, Iran) in 2017 cropping year, and chlorophyll (Chl), proline, protein, carotenoid and reducing sugars traits were evaluated. Analysis of variance showed significant differences among genotypes for the studied traits at 1% probability level. The graphical results of Hayman's analysis showed the role of over-dominance genes effects in controlling proline content, sugars content, Chl a, Chl b, total Chl and carotenoids traits whiles the protein content trait was controlled by the incomplete dominance of genes. The narrow-sense heritability for carotenoid and proline content traits were 0.14, for protein content was 0.44 and for other traits were varaied in this range. The results of this study showed that the use of heterozygosity and the production of hybrid varieties can be used to breeding traits such as proline content, sugars content, Chl a, Chl b, total Chl and carotenoids. However, for breeding of protein content, use of both methods (selection and production of hybrid) are proposed.