Background: White spotting of the coat is a characteristic trait of various domestic species including cattle and other mammals. It is a hallmark of Holstein-Friesian cattle, and several previous studies have detected genetic loci with major effects for white spotting in animals with Holstein-Friesian ancestry. Here, our aim was to better understand the underlying genetic and molecular mechanisms of white spotting, by conducting the largest mapping study for this trait in cattle, to date. Results: Using imputed whole-genome sequence data, we conducted a genome-wide association analysis in 2973 mixed-breed cows and bulls. Highly significant quantitative trait loci (QTL) were found on chromosomes 6 and 22, highlighting the well-established coat color genes KIT and MITF as likely responsible for these effects. These results are in broad agreement with previous studies, although we also report a third significant QTL on chromosome 2 that appears to be novel. This signal maps immediately adjacent to the PAX3 gene, which encodes a known transcription factor that controls MITF expression and is the causal locus for white spotting in horses. More detailed examination of these loci revealed a candidate causal mutation in PAX3 (p.Thr424Met), and another candidate mutation (rs209784468) within a conserved element in intron 2 of MITF transcripts expressed in the skin. These analyses also revealed a mechanistic ambiguity at the chromosome 6 locus, where highly dispersed association signals suggested multiple or multiallelic QTL involving KIT and/or other genes in this region.Conclusions: Our findings extend those of previous studies that reported KIT as a likely causal gene for white spotting, and report novel associations between candidate causal mutations in both the MITF and PAX3 genes. The sizes of the effects of these QTL are substantial, and could be used to select animals with darker, or conversely whiter, coats depending on the desired characteristics.