Diaporthe vaccinii causes twig blight and fruit rot and is currently listed as a quarantine organism for the European Union. In the Netherlands, two species from the same genus, Diaporthe eres and Diaporthe rudis, are regularly isolated from blighted twigs of Vaccinium corymbosum. This study compared the pathogenicity of these two species to D. vaccinii. To develop a pathogenicity test a field experiment was performed at an experimental station, testing isolates of D. rudis and D. eres. Most of the isolates tested did not cause statistically significantly larger lesions compared to the control treatment (P > 0.05). In a greenhouse experiment a similar test was conducted, comparing D. vaccinii with the closely related species D. eres. Two cultivars of blueberry, Duke and Liberty, were inoculated using two D. vaccinii and two D. eres isolates. Stem canker lesions caused by D. vaccinii were larger than these caused by D. eres isolates. The majority of the lesions caused by D. vaccinii did not pass the first node of the inoculated shoot, which might be an explanation for the ‘mild’ symptoms of D. vaccinii in V. corymbosum, which appear not to cause crop losses. The results of this study suggest that D. vaccinii may not be a major threat to blueberry production in Europe. Further data is required to be able to conclude whether the quarantine status of D. vaccinii is still appropriate.