Diaporthe ampelina (=Phomopsis viticola) is one of the most important pathogens causing both cane/leaf spot and wood canker diseases in grape growing countries in the world. In this research, morphological, molecular and pathogenic characterization of 23 D. ampelina isolates were studied and their sensitivity was tested against hot-water treatments and some of the fungicides used in vineyards. Morphologically, the isolates were grouped according to “W type” and “G type” colony appearance and microscopic features. In molecular characterization, beta-tubulin, calmodulin and translation elongation factor (tef1-α) gene regions were amplified with PCR. The nucleotide sequences were analyzed using NCBIBLAST search and recorded in GenBank, through which species identity was also confirmed. Mycelial viability was tested against hot-water treatments (46 – 50°C for 30 and 45 min) in centrifuge tubes and thermal inactivation point was determined. It was also tested against some of the fungicides (azoxystrobin, boscalid, cyprodinil, tebuconazole, azoxystrobin + cyproconazole + tebuconazole, cyprodinil + fludioxonil, azoxystrobin + tebuconazole and fludioxonil) in vitro and EC50 values were calculated. The morphological and molecular study results showed that all the isolates were D. ampelina and they were pathogenic on wood tissues of vines. Thermal inactivation of “W type” isolates was ensured at 48°C-30 min hot-water treatments. Although this treatment also reduced colony growth of “G type” isolates, it did not inhibit it completely and 48°C-45 min treatment was needed to reach full eradication. Considering fungicide sensitivity, fludioxonil or tebuconazole containing fungicides were the most effective in suppressing the mycelial growth of the fungus. However, azoxystrobin, boscalid, cyprodinil could not perform a strong inhibition when compared to fludioxonil and tebuconazole.