Fowl glioma is characterized by multiple nodular growth of astrocytes, and fowl glioma-inducing virus belonging to avian leukosis virus has been isolated from Japanese bantam as a causal agent. Subcutaneous neoplasms of the head and neck have been reported in layer chickens since 2003 in Japan, and fowl glioma concurred in these affected layers. In the present study, the histopathology of 240 layers, including 18 layers with subcutaneous neoplasms and 222 layers kept with the affected layers, was performed to clarify the characteristics of fowl glioma in layers. Microscopically, 103 layers showed non-suppurative encephalitis, and four layers had locally extensive proliferation or multiple nodules of astrocytes. Gliomas concurred in 11 layers with subcutaneous neoplasms and occurred independently in three layers. In addition, two layers had locally extensive proliferation of small, round cells in the cerebrum. The fowl glioma-inducing virus genome was not detected in the affected brains by nested polymerase chain reaction. Ten isolates were obtained from the affected brains. By nucleotide sequencing of the env gene, SU coding regions of these isolates were most closely related to myeloblastosis-associated virus-like viruses, but TM regions showed the highest similarity to endogenous viral (ev) loci. The genome of one isolate mainly consisted of ev loci and contained several parts of other avian leukosis/sarcoma viruses. These results show that the causal avian leukosis virus of fowl glioma is not just fowl glioma-inducing virus and that different avian leukosis virus strains having oncogenicity in the central nervous system by recombination are spread in layers in Japan.