Powdery (Erysiphe necator) and downy mildew (Plasmopara viticola) are two of the most economically important diseases in Australian viticulture, costing $140 million per year through reduced yields, fruit quality, and disease management (Scholefield et al. 2010). Fungicides are applied throughout the growing season to prevent and control disease. Quinone outside inhibitors (QoI) and phenylamides (e.g. metalaxyl and its isomer metalaxyl-m) are chemical groups commonly utilised for controlling E. necator and P. viticola, respectively. However, reduced efficacy of these groups has been reported (Wicks et al. 2005; Hall et al. 2017) and Sosnowski et al. (2023) recommended the removal of chemical groups from spray programs where resistance has been observed. Continued fungicide use provides a selective advantage to resistant isolates, although mutations conferring resistance may impose fitness penalties (Hawkins and Fraaije, 2018). The G143A mutation in the cytb gene (cytochrome b protein) confers resistance to QoI fungicides in E. necator, whilst the mechanism of metalaxyl resistance in P. viticola is unknown. The objective of this work is to examine the competitiveness and fitness of resistant isolates in a mixed population in the absence of the relevant fungicide. Grapevines (Vitis vinifera var. Cabernet Sauvignon) in pots were placed into mini-greenhouses (MGH) and inoculated with mixtures of QoI resistant (R) and sensitive (S) E. necator isolates in ratios of 1R:0S, 3R:1S, 1R:1S, 1R:3S and 0R:1S. Four leaves on each plant, in two separate MGH, were tagged and disease severity measured after 14 and 21 days. Healthy vines were introduced into the MGH every 21 days to allow disease transmission and consequently the creation of a new generation. Concurrently, four symptomatic leaves were collected from different regions of the diseased plant and rinsed with sterile reverse osmosis water to collect spores. The resulting suspension was subjected to high-throughput qPCR (HT-qPCR) to track the persistence of the G143A mutation after each generation (Sosnowski et al. 2023). Similarly, V. vinifera var. Sultana grapevines were inoculated with mixtures of metalaxyl-m R and S P. viticola isolates (1R:0S, 3R:1S, 1R:3S and 0R:1S). After 14 days, four leaves with downy mildew were collected and moist-incubated to advance sporulation. Leaves were rinsed to prepare a sporangium suspension, which was used to inoculate a healthy grapevine to create the next generation. A leaf disc assay (Ismail et al. 2024) was completed every third generation to track persistence of resistance to metalaxyl-m. There were two biological replicates per treatment per pathosystem. Results from molecular genetic testing for the G143A mutation will be presented. Preliminary results suggest that metalaxyl-m resistance remained stable after 11 generations on detached leaves, and six generations in greenhouse conditions. The finite number of chemical groups used for the management of E. necator and P. viticola, in addition to the labour-intensive and expensive task of developing new fungicides, emphasises the value of preserving existing ones. Understanding the persistence of resistance will assist in the design of effective spray programs, which can extend the useful life of existing fungicides.