The genetic variation in equine arteritis virus (EAV) GL protein encoding gene was investigated. Nucleic and deduced amino acid sequences from 7 different EAV isolates, including 4 eastern Canadian field isolates, were compared with those of the Bucyrus reference strain. Nucleotide sequence identities between these isolates and the Bucyrus reference strain ranged from 87.5% (Vienna isolate) to 93.9% (11958 isolate). Amino acid identities with the Bucyrus reference strain varied from 90.2% (Vienna isolate) to 95.1% (19933 isolate). A 2nd potential N-linked glycosylation site was found at position 81 in the GL protein of all EAV isolates. Three amino acid substitutions at residue position 90 (Glu-->Val), position 101 (Ala-->Val or Thr), and position 119 (Val-->Leu, Phe or Ser) were also found in all EAV isolates. Phylogenetic analysis showed that the North American EAV isolates, including the Canadian isolates, and the European prototype Vienna isolate could be classified in 2 distinct groups. Three putative sequential antigenic sites were predicted in EAV GL protein. The 1st antigenic site (TAQRFT) was located at positions 24 to 29, and the 2nd antigenic site (RYDEHTA) at positions 47 to 53. The 3rd antigenic site was predicted to be located at positions 78 to 84 and showed the less conserved amino acid sequence.