Wheat is a main food source for more than 40% of the world’s population, while pests and pathogens are widely recognized as a significant obstacle to food security. Assessing the impact of pests and pathogens on yield losses is therefore critical to maintaining global food supply. However, quantitative estimation of wheat yield losses is notoriously challenging. Here, we report on an experiment-based quantitative assessment of wheat yield losses in China caused by pests and pathogens from 2000 to 2018. We found that wheat yield losses caused by pests and pathogens in six main wheat planting regions (the Yellow and Huai River valleys, the middle and lower reaches of the Yangtze River, the Southwest China, the Loess Plateau, the Northeast China and the Xinjiang province) were 16.29%, 7.46%, 11.71%, 12.64%, 6.54% and 4.84%, respectively, of the regional yield. The proportions of yield losses caused by aphids, wheat mites, scab and sharp eyespot have been on the rise, while those of underground pests have declined. Wheat powdery mildews were shown to have increased in the Xinjiang province but declined in the Northeast China. Future breeding research should focus on varieties resistant to rising group like aphids, wheat mites, scab, sharp eyespot and powdery mildews. The greatest yield loss occurred in the Yellow and Huai River valleys which was also the place with the highest wheat yield. More efforts are needed to provide management measures on wheat health management in the Yellow and Huai River valleys. Our results provide an accurate assessment of wheat yield losses in main wheat production regions of China and help devise region-specific agricultural management strategies to secure food supply to an increasingly aging population.