The Toll-like receptor genes (TLRs) occupy key roles in the mammalian innate immune system by functioning as molecular sentries for invading pathogens. To elucidate the haplotype structure of the equine TLR genes, we utilized a next-generation sequencing approach in conjunction with downstream allele-specific genotyping assays to detect and validate 179 biallelic variants across 9 equine TLR genes for 43 established breeds (Equus caballus) as well as the Donkey (Poitou). Altogether, 35 nonsynonymous single nucleotide polymorphisms (SNPs) were detected and validated. Bayesian haplotype reconstructions and median joining haplotype networks revealed haplotype sharing between E. caballus and the Donkey at all investigated loci. Additionally, we were unable to differentiate between the specialized equine breeds (Heavy-Draft; Light-Horse; Pony) despite an average polymorphism density of 1 marker / 414 bp. Collectively, 158 tagSNPs were sufficient to predict 100% of the variation observed across all breeds. Polyphen and SIFT analyses of amino acid substitutions (AA) encoded by nonsynonymous SNPs revealed that up to 23% of the AA replacements were predicted to impact protein function. The results of this study are expected to positively augment existing equine research by providing new avenues by which to explore translational genomics and the potential for marker assisted vaccination.