Capillary zone electrophoresis of 1,948 Holstein-Friesian cows suggested the presence of an unknown protein variant of Î±-lactalbumin (Î±-LA) in the milk of 1 cow. Sequencing genomic DNA of this cow showed a polymorphism in the Î±-LA gene (LAA) that appears to be responsible for this protein variant. This single nucleotide polymorphism g.600G > T was located in exon 2 of LAA and causes the amino acid change 65Gln > His in the Î±-LA protein. This Î±-LA protein variant is a new protein variant and should be called Î±-LA protein variant D. This amino acid change is not expected to affect protein function. Genomic DNA of 156 bulls of various dairy cattle breeds was screened to examine the presence of the new Î±-LA protein variant D. Single nucleotide polymorphism g.600G > T, responsible for Î±-LA protein variant D, was not found in any of the 156 bulls. However, 10 other polymorphisms in the coding and promoter regions of LAA were detected that were used to construct haplotypes.