High-Resolution Melting (HRM) is a sensitive polymerase chain reaction-based molecular assay used to detect, single nuclear polymorphisms, mutations, and variations in genotypes of pathogens. HRM analysis is more convenient than other detection and discrimination approaches since it is a closed-tube method, that is the polymerase chain reaction amplification and subsequent analysis are carried out sequentially in the well. It finds application in various veterinary diagnoses and discrimination of genotypes of the microorganisms. Numerous public health organizations have recommended increased monitoring activities to support current surveillance programs in response to the threat of bioterrorism and emerging infectious diseases. HRM assay will be an efficient, cost-effective disease diagnostic and genotype of circulating pathogen identification method thereby aiding in disease surveillance which is important for monitoring the spread of infectious diseases. This review emphasizes the application of HRM in livestock, poultry, and companion animals in recent times carried out all over the world for determining the genotype, strain, and breed differentiation including pathogens classification.