The Smyth line (SL) chicken, an animal model for autoimmune human vitiligo, is characterized by a spontaneous posthatch pigment loss, determined to be the result of an autoimmune phenomenon. Because endogenous virus (EV) genes have been reported to be associated with a number of autoimmune diseases of human and animal models, we designed this experiment to investigate the role of EV in the SL vitiligo by using the complete sequence of Rous-associated virus-2 as a probe for EV. An F2 resource population was developed by the matings of SL and parental control (BL) chickens. Linkage disequilibrium between vitiligo and EV was apparent (16.2-kb SacI fragment, P ≤ 0.05 and a 19-kb HindIII fragment, P ≤ 0.03). Methylation analyses revealed that the EV and endogenous avian retroviral (EAV) genes were methylated in both the SL and BL sublines of chickens; therefore, methylation does not appear to be responsible for the differences in the expression of vitiligo between SL and BL sublines. Expression of the EV genes correlated with the disease in vitiliginous SL101 birds and also in 5-Azacytidine-induced vitiliginous BL101 parental control chickens. Only one EV locus was detected in the unrelated Light Brown Leghorn control chickens (1q14) by in situ hybridization, whereas 3 EV loci were identified in SL101 and BL101 chickens (1p25, 2q26, and an unidentifiable micro-chromosome). Our observations indicate that EV genes may play a role in the induction of autoimmune vitiligo in the SL chicken model.