The avian leukosis virus (ALV) is a serious threat to sustainable and economically viable commercial poultry management world-wide. Active infections can result in more than 20% flock loss, resulting in significant economic damage. ALV detection and elimination from flocks and breeding programs is complicated by high sequence variability and the presence of endogenous virus copies which show up as false positives in assays. Previously-developed approaches to virus detection are either too labor-intensive to implement on an industrial scale or suffer from high false negative or positive rates. We developed a novel multi-locus multiplex quantitative real-time PCR system to detect viruses belonging to the J and K genetic subgroups that are particularly prevalent in our region. We used this system to eradicate ALV from our broiler breeding program comprising thousands of individuals. Our approach can be generalized to other ALV subgroups and other highly genetically diverse pathogens.