Developing elite breeding lines and varieties often requires plant breeders to combine desirable traits from multiple parental lines, particularly in the case of disease resistance. The process of combining traits, known as gene pyramiding, can be accelerated by using molecular markers to identify and keep plants that contain the desired allele combination and discard those that don't. The development of molecular genetics and associated technology like Multiple Sequence Alignment (MAS) has led to the emergence of a new field in plant breeding, gene pyramiding. This term is used in agricultural research to describe a breeding approach to achieve pest control and higher crop yield. Gene pyramiding aims at developing derivation of an ideal genotype that is homozygous for the favorable alleles at all n-loci. Gene pyramiding is mainly defined as a process of combing two or more genes from multiple parents to develop elite lines and varieties. This leads to the simultaneous expression of more than one gee in a variety. When pyramiding genes, breeders must calculate the probability of an individual plant containing the desired combination of alleles. This probability dictates the population size required to have a high probability of finding at least one plant with the desired combination of alleles. Gene pyramiding is an important strategy for germplasm improvement. Pyramiding requires that breeders consider the minimum population size that must be evaluated to have a reasonable chance of obtaining the desired genotype. Molecular marker genotyping can facilitate the gene pyramiding process by reducing the number of generations that breeders must evaluate to ensure they have the desired gene combination. One of the strategies of gene stacking/pyramiding is the iterative procedure/ sexual hybridization. A plant harboring one or more transgenes is cross-hybridized with another plant containing other transgenes. Development of a multi-stack hybrid occurs via iterative hybridization. Examples of commercial stacks are maize, cotton etc. Gene pyramiding is gaining considerable importance as it would improve the efficiency of plant breeding leading to the development of genetic stocks and precise development of broad-spectrum resistance capabilities. The success of gene pyramiding depends upon several critical factors, including the number of genes to be transferred, the distance between the target genes and flanking markers, the number of genotype selected in each breeding generation, the nature of germplasm etc. Innovative tools such as DNA chips, micro arrays, SNPs are making rapid strides, aiming towards assessing the gene functions through genome-wide experimental approaches. The power and efficiency of genotyping are expected to improve in the coming decades. Information collected and the preparation of the note is assisted by Mr. Aqief Afzal, Research Assistant, ASRBC, ACI Ltd