ABSTRACTNeoscytalidium dimidiatum is an asexual, opportunistic and necrotrophic pathogen that causes the devastating stem canker disease of dragon fruit species (Selenicereus spp.). Despite this, information about its intraspecific diversity remains lacking. Examination of the morphological variations of 40 N. dimidiatum isolates hosted by S. megalanthus, S. monacanthus and S. undatus from eight allopatric regions in the Philippines detected three distinct morphotypes. Sequencing of three partial gene markers (ITS, ACT and TUB2) of representative isolates revealed low intraspecific genetic diversity of the pathogen from the three hosts. Phylogenetic analysis of internal transcribed spacer (ITS) sequences of the isolates in this study and worldwide collections of the phytopathogen in the GenBank database infers similar founding populations and the possible specificity of the dragon fruit‐infecting N. dimidiatum. Pathogenicity and aggressiveness assay of the isolates on detached stems of dragon fruit did not detect host specificity to S. monacanthus and S. undatus. However, some isolates (i.g., MBDF0802A, MBDF0808B, MBDF0339A and MBDF0007A) significantly had shorter latent period to the stems of S. undatus than S. monacanthus. A higher aggressiveness component of lesion size development on S. undatus stems by isolates from S. monacanthus was recorded suggesting a possible directional selection of highly aggressive N. dimidiatum in this host. This is the first population study on the phenotypic traits and the genetic structure of N. dimidiatum. Valuable inputs for further study of the pathogen are provided, which may help inform the design of management options for stem canker disease of dragon fruit.