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The objective of this study was to investigate whether the sensitivity of E. necator populations to several important fungicide groups has changed in Serbia. Field trials, bioassays, and molecular tests were conducted from 2019 to 2022. Samples were collected from commercial, experimental and extensive vineyards. Low efficacy of QoIs, azanaphthalenes and aryl-phenyl-ketones was observed in the field trials. A total of 110 isolates were analyzed by qPCR for the presence of the G143A mutation. The mutation was positively detected in 61.8% of the isolates, therefore isolates containing >95% of the G143A allele were QoI-resistant in bioassays. Contrary, QoI-sensitive isolates contained <5% of the G143A allele. Of the 54 isolates tested in bioassays, 46 grew significantly at the recommended concentration, whereas 36 were able to grow at a fourfold higher concentration of quinoxyfen. Proquinazid showed more significant inhibition than quinoxyfen, suggesting that there is no cross-resistance between these two members of the azanaphthalene group. Unsatisfactory effect of metrafenone and pyriofenone at recommended concentrations was observed in 29 of 41 isolates. These isolates grew readily at four times higher concentrations of metrafenone and pyriofenone, indicating the cross-resistance between them. Overall, the results of this study suggest that resistance to QoIs has developed and is widespread in commercial vineyards, requiring growers to exclude them completely from powdery mildew management. The performance of proquinazid in the field was not affected, making it suitable for powdery mildew control. Special attention and extensive monitoring should be directed to metrafenone and pyriofenone, as cross-resistance has been found at certain sites where they have been used intensively over the past decade. • Sensitivity of Erysiphe necator populations to different fungicides was studied. • Low efficacy of QoI, azanaphthalene and aryl-phenyl ketone fungicides was found. • The G143A mutation detected by qPCR analysis was prevalent in most samples. • Cross-resistance between metrafenone and pyriofenone was observed. • Proquinazid was effective for quinoxyfen-resistant powdery mildew populations. [ABSTRACT FROM AUTHOR]