The aim of our study was to detect new polymorphisms in the bovine beta-lactoglobulin (beta-LG) gene with significant effects on beta-LG protein concentration. Genomic DNA samples from 22 proven bulls were screened for polymorphisms in the coding and promoter regions of the beta-LG gene. In total, 50 polymorphisms were detected. Two single nucleotide polymorphisms (SNPs) (g.1772G>A and g.3054C>T) lead to amino acid changes and are the causal genetic polymorphisms of beta-LG protein variants A and B. Forty-two polymorphisms were in complete linkage disequilibrium (LD) with beta-LG protein variants A and B. Any of these 42 polymorphisms can be involved in the differential expression of the respective A and B alleles of the beta-LG gene. The eight polymorphisms not in complete LD with beta-LG protein variants A and B and the two polymorphisms causing the amino acid changes were genotyped in a set of 208 cows: 106 animals homozygous for beta-LG protein variant A and 102 animals homozygous for beta-LG protein variant B. Of these eight polymorphisms, six SNPs segregated only within the cows homozygous for beta-LG protein variant A and two SNPs segregated only within the cows homozygous for beta-LG protein variant B. One of the eight polymorphisms had a significant effect on beta-LG protein concentration. This SNP, g.-731G>A, segregated only within the 106 cows homozygous for beta-LG protein variant A. Within these cows, adjusted relative beta-LG protein concentration was reduced by 1.22% (w/w) in animals homozygous g.-731AA compared with animals homozygous g.-731GG.