Mutagenesis has emerged as a powerful tool in crop breeding that facilitates the creation of elite crop varieties with improved agronomic traits. Innovations in mutagenesis techniques and functional genomics have revolutionized crop improvement strategies. Various mutagenesis approaches, including chemical, physical, biological, site-directed mutagenesis, soma clonal variation and modern genome-editing techniques such as CRISPR/Cas9 are employed to generate genetic diversity for crop breeding applications. Genome level mutations result in changes in chromosome structure (such as inversions, translocations, duplications, and deletions) and chromosome number (polyploidy, aneuploidy, and haploidy), while gene mutations are point mutations in the DNA sequence. Traditional phenotyping methods and advanced genotyping techniques including PCR assay, Molecular markers, next-generation sequencing, and MutMap approaches are used to characterize these mutations. Mutagenesis serves as both forward and reverse genetic approach in crop improvement. This chapter investigates the pivotal role of mutagenesis in crop improvement and its integration with functional genomics to identify and validate candidate genes that govern important traits, including enhanced yield, better nutritional quality, resilience to biotic and abiotic stressors, and other agronomic attributes. We demonstrate how the combination of mutagenesis and genomic technologies is transforming crop breeding practices, using examples from various crop species to improve food security amid evolving environmental challenges.