The level of cytochrome b5A (CYB5A) in pig testis is correlated with boar taint from androstenone and an AF016388:c.-8G>T polymorphism in CYB5A has been linked with low androstenone levels in the fat of pigs. In this study, we developed a polymerase chain reaction-based assay to genotype 1242 boars from eight lines for the c.-8G>T SNP. The c.-8T allele was found in all eight lines at a frequency ranging from 1.8% to 20.3% with an overall frequency of 8.6%. Significant deviations from Hardy-Weinberg equilibrium were found in the Hampshire, Landrace and Yorkshire breeds. The homozygous mutant c.-8TT occurred infrequently and was not found in some lines, but was consistently associated with low androstenone levels in fat. Both CYB5A mRNA and CYB5A protein levels were decreased in the c.-8TT genotype in a subset of Yorkshire boars, suggesting that low levels of CYB5A protein in the c.-8TT mutant were not due to inefficient translation of CYB5A mRNA. There were significant but modest marker effects on fat androstenone levels in Landrace, Yorkshire and a Large White/Duroc cross and fat skatole in Duroc and Sire Line breeds. There was no effect of CYB5A genotype on bulbourethral gland length, suggesting that this SNP will not affect reproductive traits. We conclude that the c.-8G>T SNP in the CYB5A gene has a significant but modest effect on boar taint in male pigs and could be useful in some breeds as part of a panel of SNP markers in a marker-assisted selection programme to produce low boar taint pigs.