Soiled-bedding sentinels are not always efficient in detecting pathogens in rodent colonies. In this context, PCR-based testing can be more sensitive and is being advocated as adjunct to traditional health-monitoring methods. PCR testing of biological samples is commonly used to confirm positive results from sentinels or detect pathogens in quarantine. Intra-cage environmental PCR is sensitive in detecting some agents but requires large sampling sizes to be representative and can be costly and unrewarding, especially when pathogen prevalence is low. Another strategy is to sample exhaust air dust from ventilated racks where particles accumulate to detect pathogens at the rack level. This strategy has proven useful in detecting fur mites, MHV and Sendai virus, but results were less conclusive for other agents. Overall, optimal sampling strategies for environmental PCR have not been established for different microorganisms. As importantly, PCR testing may yield false-positives by amplifying nonspecific DNA or identifying genetic material in the absence of infectious particles. Our group was one of the first to raise concerns on environmental PCR following false-positive pinworm results due to non-specificity of commercial assays and amplification of Rhabditid nematodes in bedding. In this presentation, we will build on our past experience with environmental PCR in dealing with pathogen outbreaks and will describe current strategies on these new diagnostic assays. We will review the methodology used by reference laboratories to validate PCR assays and discuss the necessary controls to integrate these molecular methods efficiently and economically in rodent health monitoring programs.