The wood decay fungi Cytospora sorbicola and Calosphaeria pulchella severely threaten the worldwide cultivation of sweet cherry trees (Prunus avium L.). Both fungi cause similar symptoms, including vascular necrosis, which leads to branch and twig dieback. In advanced stages of the disease, cankers are visible on tree branches and trunks. The sweet cherry is the most widely planted fruit tree in Chile, with 74,000 hectares in 2023. According to the planted surface, the predominant sweet cherry varieties are Lapins, Santina, Regina, and Bing. Variety-dependent susceptibility studies on Cyt. sorbicola and Cal. pulchella are lacking. The main entry points for wood necrosis-causing fungi are pruning wounds; therefore, we evaluated the aggressiveness of Cyt. sorbicola and Cal. pulchella in one-year-old sweet cherry plants. Santina and Lapins showed the lowest necrotic lesion caused by Cyt. sorbicola (13.6 and 14.31 mm, respectively), followed by Bing (19.51 mm) and Regina (26.14 mm). All plants infected by Cyt. sorbicola showed shoot blight regardless of the variety. In addition, there was a varying susceptibility to Cal. pulchella, with Lapins (21.6 mm), Bing (22.83 mm), Santina (27.62 mm), and Regina (30.8 mm) showing increasing levels of observed necrosis. The lesion caused by Cal. pulchella was more significant than that observed for Cyt. sorbicola, regardless of the cherry tree genotype. We identified each fungal growth from the wood necrosis progression area using two independent novel PCR-HRM strategies based on the ITS fungal region, which allowed us to differentiate each pathogen of interest individually or simultaneously. This study demonstrates different levels of susceptibility of sweet cherry tree genotypes to wood-degrading pathogens, emphasizing the need to include these factors in phytosanitary management programs.