Global human population growth, climate change and the limitation of fossil fuels will put new human pressure on food, medicine, fuel and food supplies in the 21st century. In order to get out of this constraint, increasing the production of higher-yielding crops of calories, starches, foodstuffs, natural pharmaceutical compounds and other important products can be a breakthrough. For this purpose, natural and induced mutations can be used to produce generations with superior and inherited properties. Several methods have been used to understand the function of genes such as RNA interference; gene scilencing and site directed mutagenesis. All these techniques require transgenesis which is not always possible in many important commercial crops. In this paper, an effective and non-transgenic approach called TILLING is discussed. This method is a reverse genetic method with high, fast and low cost ability that determines the allelic series of induced point mutations in the desired genes in the population of the chemically/physically mutated individuals. The main advantage of TILLLING is its applicability to any species, regardless of ploidy level and genome size. The vast applications of this powerful technique in applied and basic research have been done through the optimization of the original TILLING strategy, such as EcoTILLING and De-TILLING.