The distribution of endogenous virus (ev) genes was studied in five commercial layer lines; four were brown-egg types and one was White Leghorn. The DNA samples were obtained from nine birds of each line and digested with SacI and BamHI endonucleases. The DNA fragments were separated by gel electrophoresis, and Southern blots were prepared and examined for the presence of ev genes following hybridization with the labeled recombinant plasmid pRAV-2 and autoradiography. Almost all fragments were present in more than one line, suggesting that each line has drawn a random assortment of ev genes from the same common pool, possibly as a founder effect. A great degree of polymorphism is shown by the ev genes in the lines investigated. Most fragments were present at low or intermediate frequencies. Very few fragments were present in only one line or in only one bird. This suggests that de novo integration of ev genes at new sites is a relatively rare event. Very few fragments were present at high frequencies, and none of the fragments, except for the BamHI internal fragments, were present in all birds of all lines. Considering that ev genes are present in the wild progenitor of the domestic chicken, the lack of fixation of any ev genes in the chicken genome is remarkable. It is proposed that ev genes have general deleterious effects in the chicken (hence, the lack of fixation), but those ev genes that have been retained in the chicken genome have favorable effects under some circumstances with consequent equilibrium at low to intermediate frequencies. It is speculated that the retained ev genes may represent sites of favorable mutation via insertional mutagenesis and, hence, a potential route to the cloning of genes of economic importance in poultry.