Eutypa lata is a fungal pathogen that causes Eutypa dieback, infecting grapevines (Vitis spp.) through wounds and colonising the vascular tissue, causing necrosis and eventually vine death (Carter 1991). For research and evaluation, the presence of E. lata in grapevine has traditionally been assessed by cutting pieces of wood from the margin between discoloured and normal tissue, placing them on potato dextrose agar (PDA) and observing the morphological growth of cultures after 7 days (Carter and Moller 1977, Sosnowski, Creaser et al. 2008). It is a well-established technique used worldwide, but it is time consuming, costly and results can be hindered by contamination with other organisms or misidentification. With the aim of developing a more sensitive, reliable and consistent technique for assessing E. lata infection, a quantitative polymerase chain reaction (qPCR) assay for E. lata was developed. Utilising existing work on Eutypa dieback qPCR assay design by Sosnowski et al. (2017), a TaqMan qPCR assay targeting E. lata was developed in the ITS region. The assay was validated against six E. lata isolates and thirty-two off-target species, mainly plant pathogens within the Dothideomycetes and Sordariomycetes classes. The assay was sensitive, and only failed in specificity testing against Diatrypella vulgaris, another pathogen implicated in Eutypa dieback. The qPCR assay was compared with the culture method using an existing vineyard trial. The trial included various treatments and positive and negative controls, and all wounds, except for negative controls, were inoculated with approximately 200 spores of E. lata on the day of pruning. One year later, the canes were collected from the vines then cut in half longitudinally after removing the bark and desiccated stub. Half of the canes were assessed by isolation and the other half via qPCR. Isolations were conducted on PDA as described by Sosnowski et al. (2008). For qPCR, crude DNA extracts were prepared by grating 100 mg of normal tissue just below the margin of discoloured wood in 1 mL of a solution containing 2% (w:v) polyvinyl pyrrolidone and 0.2% (w:v) sodium diethyl dithiocarbamate dissolved in PBS buffer, and beating in a tissue lyser (Qiagen) for 2 minutes at 24 bpm. Five ml of the plant crude extracts were used as template and analysed in triplicate by qPCR. Of the 638 samples that were analysed, 374 corresponded for both methods, resulting in 59% of coincidental results and a Cohen’s kappa index of 0.2245 ± 0.0281, indicating ‘fair agreement’ (Cohen 1960, Landis and Koch 1977). A further 252 samples were positive for qPCR but not isolation, and only 12 were positive for isolation but not qPCR. These results showed that qPCR was considerably more sensitive than isolation for detecting the presence of E. lata in experimental grapevine canes, offering a more accurate, rapid and reliable method. qPCR assessment is however 1.5 times more expensive that the isolation method. Further evaluation of the qPCR method is planned to investigate reducing replication and confirming accuracy of the method for wound susceptibility experiments.