White spotting patterns in mammals can be caused by mutations in the gene KIT, whose protein is necessary for the normal migration and survival of melanocytes from the neural crest. The alpaca (Vicugna pacos) blue-eyed white phenotype (BEW) is characterized by two blue eyes and a solid white coat over the whole body. Breeders hypothesize that the BEW phenotype in alpacas is caused by the combination of the gene causing grey fleece and a white-spotting gene. We performed an association study using KIT flanking and intragenic markers with 40 unrelated alpacas, of which 17 were BEW. Two microsatellite alleles at KIT-related markers were significantly associated (P < 0.0001) with the BEW phenotype (bew1 and bew2). In a larger cohort of 171 related individuals we identify an abundance of an allele (bew1) in grey animals and the occurrence of bew2 homozygotes that are solid white with pigmented eyes. Association tests accounting for population structure and familial relatedness are consistent with a proposed model where these alleles are in linkage disequilibrium with a mutation or mutations that contribute to the BEW phenotype and to individual differences in fleece color.