Different DNA markers have been utilized in the last few decades as important molecular tools in plants for genetic relation studies among individuals, hybrid and varietal identification, phylogenetic relationship among species, gene mapping and tracking quantitative trait loci. These markers can be broadly classified into hybridization and PCR based markers. Restriction fragment length polymorphism (RFLP) represents the hybridization based marker; while PCR dependent includes more reliable and advanced polymorphic markers like amplified fragment length polymorphism (AFLP), inter-simple sequence repeats (ISSR), single nucleotide polymorphism (SNP), sequence related amplified polymorphism (SRAP), start codon targeted (SCoT) and inter-primer binding site (iPBS) among others. Functional markers (FMs) have also been developed from functionally characterised sequence motifs which are superior to random markers due to their complete linkage to trait locus alleles. With the advent of next generation sequencing (NGS) technology, excellent opportunities are offered for generating ample structural and functional genomic information in many important crops. The novel approach of genotyping-by-sequencing (GBS) employs NGS protocols for discovering and genotyping SNPs in many important crop genomes and populations. The present review focuses on the description of varied molecular markers, their methodologies, strengths and limitations as well as applications in plant breeding and genetic research.