Genetic analysis of the kappa-casein gene (CSN3) promoter regions of 42 cattle representing 9 different breeds revealed that 2 distinct haplotypes (A and B) exist at this locus, differing from each other by single base changes at positions -514 (T/G), -426 (T/C), and -384 ( T/C), where haplotype A has bases T, T, and T and haplotype B has bases G, C, and C. The AA and AB haplotypes were found to occur at a higher frequency in the animals tested, with 69.0 and 21.4% being homozygous and heterozygous, respectively. The sequences that include these polymorphisms are potentially important in transcriptional regulation of the kappa-casein gene, because they contain putative sites for binding of many transcription factors. Linkage disequilibrium between the kappa-casein promoter haplotype and either one of the 2 major kappa-casein coding sequence haplotypes was not evident. The A allele is dominant in all groups (dairy, beef, and dual purpose) with an allele frequency of 80% and is higher among high-yielding dairy animals (88.9%) than among beef animals (75%). The AB haplotype is comparatively rare in the dairy cattle (11.1%) compared with both beef and dual-purpose animals. The BB haplotype, though rare overall (9.5%), is much higher in dual-purpose animals (18.8%) than dairy (5.6%) animals. In contrast, the B allele is much more representative of the kappa-casein promoters from other ruminants.