The prolactin receptor (PRLR) is a receptor for a polypeptide hormone that stimulates mammary gland development and lactation in mammals. Associations between the PRLR genotype and cow and goat milk traits have been reported. To date, the nucleotide polymorphisms within the PRLR gene in equine species have not been investigated. Single-nucleotide polymorphism (SNP) is the most abundant form of genetic variation and a resource for useful genetic traits. In the present study, SNPs within the coding regions of the PRLR gene were detected by polymerase chain reaction-single-strand conformation polymorphism (SSCP) and DNA sequencing on the Yili horse of China. Ten novel SNPs were identified in the PRLR gene: one located within exon 4 (XM_001500104: c.156 C>T); one within exon 5 (c.312 T>C); and eight within exon 10 (c.1062 T>C, c.1083 G>A, c.1226 A>C, c.1233 C>T, c.1279 C>A, c.1749 A>T, c.1764 G>C, and c.1778 T>A). The mutation allele frequencies and effective allele numbers ranged from 0.10 to 0.30 and from 1.2195 to 1.7240, respectively. Four of the identified SNPs are non-synonymous and involve four amino acid substitutions, namely, p.H409P (c.1226 A>C), p.H427N (c.1279 C>A), p.Q588H (c.1764 G>C), and p.I593K (c.1778 T>A), in the cytoplasmic domain of the PRLR, which plays a fundamental role in signal transduction. Theoretically, the four nucleotide substitutions would result in altered spatial structure and functions of the PRLR molecules. Further study is required, however, to determine whether these mutations affect the milk yield of the Yili horse.