The large-conductance, Ca-dependent K channel plays a key role in the control of vascular tone. Variation in the gene encoding the beta-1 subunit of the Ca-dependent K channel (KCNMB1) has been reported to be associated with hypertension, however, variants in KCNMB1 have not been systematically characterized to date. In this study, we have performed the most comprehensive evaluation to date of single nucleotide polymorphisms in KCNMB1 using genomic DNA from 60 individuals of European, African and native American ancestry. We identified and characterized single nucleotide polymorphisms in the exons, intron/exon junctions, upstream region and 3' untranslated regions of KCNMB1 using denaturing high-performance liquid chromatography combined with direct DNA sequencing. A total of 25 single nucleotide polymorphisms in KCNMB1 were identified. Seven of the polymorphisms (28%) are novel single nucleotide polymorphisms not reported previously. Allele frequencies range from less than 1.7 to 50% and 19 single nucleotide polymorphisms had a minor allele frequency greater than 5%. A lack of strong linkage disequilibrium among the 25 single nucleotide polymorphisms was observed in all three race/ethnicity groups; therefore the identification of haplotype 'tag' single nucleotide polymorphisms for genetic association studies is not likely to be appropriate for KCNMB1. Multiple species comparative analysis and in-silico functional analysis were performed to identify potential functionally important single nucleotide polymorphisms within the gene. These data highlight that a tag single nucleotide polymorphism approach will not be appropriate for the study of genes such as KCNMB1, although potentially important functionally significant single nucleotide polymorphisms are suggested for future studies investigating the influence of this gene's variability on disease and drug response.