A BamHI, EcoRI, and XhoI restriction endonuclease map of Marek's disease virus (MDV) serotype 2 (MDV2) DNA was constructed by double-digest analyses of 28 cloned BamHI and 11 cloned EcoRI fragments of MDV2 DNA, followed by hybridization tests of these cloned BamHI DNA fragments with electrophoretically separated digests of MDV2-infected cell DNA. On this map, MDV2 genome consisted of two segments which have unique regions inserted between two inverted repeat regions as observed in MDV serotype 1 and 3 genomes. Further, the DNA homology among three serotypes of MDV was examined by hybridization under less stringent conditions using cloned BamHI fragments of MDV2 DNA. Most of the MDV2 fragments located within the unique regions hybridized with MDV serotype 1 and 3 DNAs, indicating the presence of the collinear relationship among three serotypes. In addition, MDV2 DNA fragments which hybridized with the DNA fragments encoding MDV1 gp57-65 (or A antigen) or MDV1 gp100, gp60, gp49 (or B antigen) were identified and these fragments of serotypes 1 and 2 found to be collinear.