The parasitic nematode Angiostrongylus vasorum is an emerging challenge for companion animal and wildlife health, with reported increases in both distribution and incidence in Europe. To facilitate improved detection of this parasite, a SYBR green real-time polymerase chain reaction (PCR)was developed to amplify a region of the second internal transcribed spacer (ITS-2) of A. vasorum from both definitive and intermediate host samples. The PCR assay was capable of detecting a single of plasmid DNA containing the entire ITS-2 region, a single first stage larva (L1) in 200 ml canine EDTA blood, a single L1 in 200mg of canine faeces and a single L3 in 10mg of Biomphalaria glabrata tissue. The assay exhibited a high level of specificity to A. vasorumandwhilst it potentially amplifies DNA of other Angiostrongylus species, it did not amplify DNA from a range of other common canine parasitic nematodes. Field evaluation of the PCR assaywas conducted by screening canine EDTA blood and faecal samples from suspected cases of A. vasorum infection and compared with Baermann’s detection, and also by screening a range of gastropod species from an endemic area. Real-time quantitative PCR offers a more efficient means of detecting A. vasorum infection with a lower limit of detection than traditional diagnostic tests, and it therefore has important clinical and epidemiological applications.