In order to develop an assay to distinguish the infection of Equine infectious anemia virus (EIAV) American strains from Donkey-leukocyte attenuated virus (DLV) strain, eight primers were designed based on the comparison of complete sequences of four EIAV American strains and DLV. Corresponding viral genome fragments were amplified and analyzed from donkey leucocytes by PCR using these eight primers. Four primers from gag gene were selected to detect the differences between EIAV American strains and DLV. Six horses were inoculated with DLV and two with EIAV American strains, the Wyoming strain and the Argentina strain, respectively. Two fragments, 675bp and 400bp, were amplified from DLV-infected cells, while only one segment of 675bp was obtained from cells inoculated with American strains. Our results indicate that this nest multiplex PCR can be used to distinguish EIAV American strains from the donkey leucocyte-attenuated vaccine strain of EIAV.