To determine the use of measuring of (1→3)-β-D-glucan for diagnosis of systemic mycosis, we conducted a retrospective analysis. We investigated the clinical records of febrile patients whose blood samples were positive for Candida or Trichosporon or who were diagnosed with invasive aspergillosis based on autopsy. Five patients were diagnosed as infected with Candida species. Although 2 cases were initially diagnosed as negative because of a low level of (1→3)-β-D-glucan at the time of the first symptom, it became elevated after 2 or 3 days during the febrile episode, as it did in all 2 patients with infection of Trichosporon asahii. One patient was diagnosed with invasive aspergillosis. (1→3)-β-D-glucan remained below the cut-off value (20 pg/mL) throughout the febrile episode. Only 2 out of 8 patients had positive results at onset of fever, whereas 7 out of 8 patients had at least 1 positive result during episode. Measurement of (1→3)-β-D-glucan antigenemia produces a low ratio of pseudonegative cases for patients with systemic mycoses and is thus useful for the diagnosis of this fungal infection.