The canine hookworms Ancylostoma braziliense, Ancylostoma ceylanicum, Ancylostoma caninum and Uncinaria stenocephala are not only capable of producing morbidity and mortality in dogs but are also neglected tropical zoonoses. Each hookworm species differs considerably in its geographical distribution, life cycle, biology, pathogenic impacts on both canine and human hosts, zoonotic potential and response to treatment with anthelminthics. Here we describe the development and validation of two Taq-Man based multiplex PCR assays capable of detecting and differentiating all four canine hookworm species in faeces of naturally infected dogs. The analytical sensitivity of both assays was assessed using 10-fold serial dilutions of synthetic gene block fragments containing individual sequence targets of each hookworm species. The sensitivity of the assays and ability to detect mixed species infections were compared to a conventional PCR-Restriction Fragment Length Polymorphism based-approach when applied to laboratory and field samples from endemic areas. The qPCRs detected at least one species of hookworms in 82.4% of PCR-RFLP-negative but microscopy-positive samples. The qPCRs detected an additional 68% mixed infections with different species of canine hookworms, and additional single species infection with A. caninum (47%), U. stenocephala (33%) and A. ceylanicum (0.02%) that were missed by PCR-RFLP. These multiplex qPCR assays will assist field based epidemiological surveillance studies towards an accurate and sensitive monitoring of canine hookworm infections in dogs, to inform their species-specific zoonotic risks to populations living in endemic areas, globally. Author summary: There are four species of blood-sucking hookworms that infect dogs, namely Ancylostoma braziliense, Ancylostoma ceylanicum, Ancylostoma caninum and Uncinaria stenocephala. Each hookworm species differs in its geographical distribution, impact on canine health and more importantly its ability to infect and produce disease in humans. For example, dogs are the primary reservoir for A. ceylanicum, the second most common hookworm species infecting humans in the Asia Pacific. Ancylostoma braziliense is more geographically confined and responsible for chronic skin eruptions in humans. Accurate diagnosis of hookworm species in dogs is therefore essential for understanding the relative risk each species pose to human populations living in close association with dogs. As microscopic methods are unable to distinguish between species of hookworms in stool, highly efficient real time PCR assays capable of detecting and differentiating all four canine hookworm species in canine faeces were developed. These new assays are superior to previously published gel-based PCR assay displaying higher throughput, and ability to correctly identify single and mixed hookworm species infections with higher accuracy. [ABSTRACT FROM AUTHOR]