A highly virulent strain of equine infectious anemia virus (EIAV) lost its fatal virulence but retained the desired antigens during serial passage over 130 generations in leukocytes in vitro. We compared the long terminal repeat (LTR) sequences of the different generations and found that three stable genetic variations occurred in the transcriptional start site, the initial base of TAR, and the pre-mRNA cleavage site at the R-U5 boundary, respectively. These three mutations happened at the inflexion of virus pathogenicity loss; therefore, the function of these mutations needs to be further addressed.