The polymorphism of the canine major histocompatibility complex class II DRB gene, DRBB1, was analyzed. Exon 2 that encodes the polymorphic beta 1 domain was amplified by the polymerase chain reaction (PCR). The PCR product from 250 dogs was cloned and sequenced. Eighteen alleles were identified. Most of the variation in amino acid composition occurred at positions in the peptide binding site. Inheritance of these sequences showed Mendelian segregation with one or two alleles per dog. Cluster analysis of the nucleotide and predicted amino acid sequences subdivided the canine DRBB1 alleles into three major allelic groups. The number of nonsynonymous changes was higher than the number of synonymous changes in the putative antigen recognition sites indicative of positive selection. The data generated can serve as a basis for developing a typing assay for the canine DRBB1 gene.