Highbush blueberry (Vaccinium corymbosum L.) is becoming an important berry fruit in Serbian agriculture, and the areas under blueberries are rapidly expanding. Since 2011, blueberry bushes with the symptoms of twig dieback and shoot canker were noticed in four important producing regions in the country. To identify the causal agent, 40 symptomatic bushes from four districts were sampled and analyzed. Based on morphological characteristics, all analyzed isolates were preliminary identified as Neopestalotiopsis clavispora. Selected isolates were further characterized by sequence analysis of the rDNA internal transcribed spacer region, translation elongation factor 1-alpha, and β-tubulin (TUB). Sequence and phylogenetic analysis confirmed that all isolates belong to N. clavispora. The pathogenicity tests on blueberry ‘Elliot’ confirmed that tested isolates were pathogenic. The detection of N. clavispora, a causal agent of twig dieback on blueberries in Serbia, is the first report of this pathogen in the country.