The zebrafish, Danio rerio, is an ideal model organism to address a broad array of biomedical questions, and is now widely employed to answer questions about aging, behavior, cancer, growth, immunity, infection, metabolism and toxicology, using adult fish as well as embryos. However, the rapid expansion of the zebrafish as a model organism has outpaced the design and implementation of sentinel health monitoring programs in zebrafish colonies. Unlike rodent facilities, relatively few zebrafish facilities have health monitoring programs in place. Several studies now document the confounding effects of subclinical disease in zebrafish, and extensive evidence from other animal models demonstrates that both clinical and subclinical naturally-occurring infections can result in invalid or confounded experimental results. Improvement in the health monitoring of zebrafish colonies is therefore an important objective in zebrafish facility management. Health monitoring for zebrafish colonies historically relied on traditional methods, such as histopathology and wet mounts. Recently, however, molecular diagnostic assays have been developed for multiple pathogens, including five parasites, Pseudoloma neurophilia, Pseudocapillaria tomentosa, Piscinoodinium pillulare, Pleistophora hyphessobryconis, and Ichthyophthirius multifiliis, four bacterial pathogens, Edwardsiella ictaluri, Mycobacterium chelonae, Mycobacterium haemophilum, and Mycobacterium marinum, and one emerging virus with a broad host range, Infectious spleen and kidney necrosis virus (ISKNV). Clinical validation studies comparing the molecular assay results with traditional diagnostic methods demonstrated that molecular diagnostic assays were more sensitive than traditional methods. The availability of sensitive molecular diagnostic assays enables laboratory animal veterinarians, facility managers, and investigators to design health monitoring programs which prospectively utilize both diagnostic modalities to improve health monitoring for zebrafish colonies.