With the advent of affordable DNA sequence technologies, large amount of genome sequences of various plant species is available which have opened new possibilities for the use of molecular biology tools and techniques in crop improvement. In crop improvement programmes Major problem faced by the breeders is the narrow genetic base of available lines and varieties which invalidate the efforts for increasing productivity and also increases the vulnerability towards biotic and abiotic factors. To widen the genetic base of any crop mutation breeding technique has been used in plant breeding since decades. It has also been used in genetic studies to identify a specific gene and to link mutation to phenotypic change in mutated organism led to the discovery of induced mutation. In the present era with the accumulation of large-scale sequence data traditional practices of plant breeding have been combined with modern biotechnological tools to elucidate the biological meaning or phenotype to genic sequence data and can create new varieties. New techniques do not replace the traditional practices; they just increase the efficiency of conventional breeding. TILLING (Targeting Induced Local Lesions IN Genomes) is one such technique which integrate the traditional mutation breeding and biotechnological methods like single nucleotide polymorphism discovery methods to identify the function of a particular gene. In functional genomics TILLING is a reverse genetics approach in which mutations have been induced in the specific gene sequence which create nucleotide sequence variation in that particular sequence which further assayed for phenotype to elucidate the gene function. As biological tools for the comparison of sequence provides sufficient information to study a gene of interest. There are screening techniques available in modern crop science that can detect protein sequence similarity to previously annotated genes thus allowing a related function to be inferred. Detection of a gene function is primarily done by reverse genetics instead of forward genetics. It is based on the change of a gene structure following analysis of the associated alteration in plants. Several other reverse genetics technologies, such as insertional mutagenesis with TDNA, transposon/retrotransposon tagging or gene silencing using RNA interference, have also been proposed for plant functional genomics. However, all the above cited methods are applicable to the model plants with small genomes and also in these species are some drawbacks that limits their utilization. Since, the discovery of TILLING technique makes it of high adoption due to its applicability across species even in plant kingdom inspite of ploidy level and genome size of an individual. This technique provides high frequency of point mutations randomly distributed in genome. Chemical mutagens have great potential towards nucleotide substitutions. Rapid and low cost identification of new alleles in a mutated population could be possible through high throughput TILLING. Diversification of bioinformatic tools, new methods including discovery of SNP using next generation sequencing are the basis of recent trends in TILLING. Wider applications of this throughput method in basic and applied research have already been implemented through modifications of the original TILLING strategy, such as iTILLING, Ecotilling or Deletion TILLING.