To evaluate the impact of clinical, histopathological, and molecular biological parameters on the prognosis of nasopharyngeal carcinoma (NPC). The clinical records of 48 patients with a diagnosis of NPC were retrospectively reviewed. In situ hybridization for Epstein-Barr virus encoded small RNA 1 (EBER-ISH) was applied for 30 paraffin-embedded specimens available. Prognostic factors of NPC were meticulously analyzed. The EBER-ISH signal was shown to be highly significant as a favorable prognostic factor (P = 0.007). Although EBV was more commonly associated with Type III NPC, EBER-ISH-positive Type I-II NPC had also significantly better survival rate than EBER-ISH-negative Type I-II NPC (P = 0.036). In addition to the WHO histopathological grade, the 1997 UICC staging, nodal status, and distant metastasis at presentation, the EBER-ISH signal was shown to be significant as a prognostic factor. This is the first report to describe the EBER-ISH as an independent prognostic factor of NPC regardless of histopathology. EBM rating: B-2b