The rapid growth of aquaculture production and international trade in live fish has led to the emergence of many new diseases. The introduction of novel disease agents can result in significant economic losses, as well as threats to vulnerable wild fish populations. Losses are often exacerbated by a lack of agent identification, delay in the development of diagnostic tools and poor knowledge of host range and susceptibility. Examples in bluegill sunfish (Lepomis macrochirus) and the giant guitarfish (Rhynchobatus djiddensis) will be discussed here. Bluegill are popular freshwater game fish, native to eastern North America, living in shallow lakes, ponds, and slow moving waterways. Bluegill experiencing epizootics of proliferative lip and skin lesions, characterized by epidermal hyperplasia, papillomas, and rarely squamous cell carcinoma, were investigated in two isolated poopulations. Next generation genomic sequencing revealed partial DNA sequences of an endogenous retrovirus and the entire circular genome of a novel hepadnavirus. Giant Guitarfish, a rajiform elasmobranch listed as vulnerable on the IUCN Red List, are found in the tropical Western Indian Ocean. Proliferative skin lesions were observed on the ventrum and caudal fin of a juvenile male quarantined at a public aquarium following international shipment. Histologically, lesions consisted of papillomatous epidermal hyperplasia with myriad large, amphophilic, intranuclear inclusions. Deep sequencing and metagenomic analysis produced the complete genomes of two novel DNA viruses, a typical polyomavirus and a second unclassified virus with a 20 kb genome tentatively named Colossomavirus. The goals of this research were to: 1) describe the various lesions in fish and associated viral agents by light and electron microscopy; 2) characterize the agents using molecular techniques and investigate their evolutionary phylogeny; 3) develop methodologies to rapidly detect each agent; and 4) determine the presence of these agents in available contact animals. Accomplishment of these objectives has provided data on pathologic changes associated with four novel viruses in fish, molecular and phylogenetic characterizations of the agents and diagnostic protocols for their detection.