Gladiolus mutants developed using gamma irradiations and EMS treatments were characterized using morphological, genetic and Polymerase Chain Reactions (PCR) based Inter-Simple Sequence Repeats (ISSR) for designing future breeding strategies. As many as 7 mutants based on phenotypic variations such as colour and shape modifications were subjected to molecular characterization using 11 ISSR primers that confirmed variations. ISSR markers showed 13.88 to 36.00 percent polymorphism among the mutants was recorded, signifying variability among different mutants. UPGMA (unweighted pair group method with arithmetic mean) based analysis of cluster separated gladiolus variety prince of orange mutants into four clusters. ISSR markers were subjected for comparative analysis like effective multiplex ratio, marker index, polymorphic information content and resolving power wherein, highest EMR (3.24), MI (1.32) were found for UBC 810, Rp (4.37) values were found for UBC 827, and maximum PIC value (0.46) for ISSR 22218 UBC 812. The results clearly indicated the reproducibility of ISSRs and their ability to detect variability among the mutants. A significant level of genetic variation was depicted by ANOVA which suggested the usefulness of studies in gladiolus breeding and unique bands obtained could be utilized for mutant/ varietal identification.