SNPs are fast becoming the marker of choice in population genetics studies of model organisms. However, a lack of efficient means to assay sufficient numbers of SNPs in non-model organisms has prevented their widespread use for conservation-related applications. We established a SNP genotyping assay (including development of new SNP markers) for detection of genetic variation in coding and non-coding parts of the fox genome, using a 96-locus multiplex format. Coding SNPs were associated with Agouti and Mc1r coat-color alleles, phylogenetically basal mitochondrial mutations, sex markers, B-chromosome protooncogenes, and serotonin-receptor genes. Non-coding SNPs (n = 51) included 27 newly described microsatellite-linked SNPs. A panel of red foxes (n = 96) was used to characterize minor allele frequencies and Hardy-Weinberg equilibrium. Genotyping accuracy was 99% for 72 SNPs relative to sequenced and known-phenotype animals.Keywords Coat-color Á Red fox Á Single nucleotide polymorphisms Á SNPs Á Vulpes vulpes Single nucleotide polymorphisms (SNPs) occur in coding and non-coding regions of the genome and are, in principle, the ideal marker for population genetics studies (Morin et al. 2004). However, because SNPs are typically biallelic, large numbers of markers are required for most applications, which has thus far prevented their widespread application in studies of non-model species, including most threatened and endangered species. While various approaches have been successfully used to discover moderate numbers of SNPs in non-model organisms (e.g., Sacks and Louie 2008), published genotyping assays used in conservation-related applications rarely involve more than a dozen or so multiplexed markers. As part of a broader research project aimed at conservation of threatened red fox (Vulpes vulpes) populations, we developed several new SNP markers and a multiplex assay for genotyping 96 coding and non-coding SNPs in red foxes. Here, we describe these markers along with the assay and its validation.We used the Golden Gate platform and BeadXpress reader (Illumina, Inc., San Diego, California), which supported genotyping assays of up to 96 SNPs per well and a 96-well format (9,216 reactions;Fan et al. 2006). Sequences containing SNPs were submitted to Illumina's proprietary Assay Design Tool (http://www.illumina.com), and a subset of 96 loci was selected based on the manufacturer's design Electronic supplementary material The online version of this article