A case of aspergillosis in 39-day-old layer chickens having a history of gradual emaciation and subsequently death with nervous signs such as torticollis and lack of equilibrium was documented. Based on the results from serology and polymerase chain reaction (PCR) test, this flock was not affected with known viral or bacterial diseases. On postmortem examination of the affected birds, multiple white to yellow nodules measuring 1~5 mm in diameter were observed in the lungs, cerebrum, liver and kidney. Microscopically, these nodules were identified as granulomatous lesions characterized by mixed population of multinucleated giant cells and lymphocytes. By periodic acid-schiff staining and nucleotide sequencing analysis, Aspergillus flavus with characteristic septate and branched hyphae were identified in the granuloma of lung and cerebrum. This case was a chronic and multisystemic aspergillosis specialized to central nervous system caused by Aspergillus flavus infection in the layer flocks.