New blueberry plantings in South Africa have increased dramatically since the year 2000, exceeding the global expansion rate. The crop is, however, impacted by several economically important fungal pathogens. Lesser-known fungi such as pestalotioid species can cause leaf and twig blight, and have the potential to become a global threat to blueberry production and expansion. This study aimed to assess the presence of such fungal species in blueberry nurseries in South Africa due to the industry's exponential growth and mass introduction of new cultivars. Symptomatic leaf and twig samples were collected from six propagation nurseries, resulting in 180 pestalotioid isolates, of which a subset of 48 isolates were selected for molecular characterisation using multi-locus sequence analysis of the internal transcribed spacer region (ITS), partial translation elongation factor 1-α ( TEF1 ) and partial β-tubulin ( TUB2 ) gene regions. Phylogenetic analyses were performed using Maximum Likelihood, Maximum Parsimony and Bayesian Inference methods. Based on the phylogenetic analyses, all isolates clustered with three previously described species of Neopestalotiopsis , namely N. rosae , N. hispanica and N. longiappendiculata. Of these, 32 isolates were identified as N. rosae, followed by N. hispanica (12) and N. longiappendiculata (4) . To the best of our knowledge, this is the first report of these species in South Africa. Based on phylogenetic analysis and morphological comparisons, we further recommend that N. vaccinii be considered a synonym of N. hispanica.