Genetic polymorphisms in genes encoding milk proteins are of interest to the animal breeding and dairy industry due to their effects on production traits, milk composition and milk quality. For this purpose, the total length of the CSN1S1 gene has been sequenced and characterised in Assaf animals with an extreme phenotype for milk protein content. In the present study, some of the polymorphisms were genotyped for association analysis studies with milk protein content in a population of 444 Assaf ewes. Furthermore, the influences of these polymorphisms on the transcription rate of this gene were also evaluated in 45 Churra Tensina ewes. An 18427-bp DNA-sequence of the CSN1S1 gene, including the entire CDS, 5'- and 3'UTR regions, and the promoter region, was analysed, leading to the identification of 61 polymorphisms. Two polymorphisms detected in the 5' flanking region were located within possible trans-acting factor binding sites, modifying the putative CdxA and GATA-1 consensus sites. The SNP (JN560175 g.1123C>A) that modifies a putative CdxA consensus site showed a significant effect on the expression of the CSN1S1 gene (P=0.043), but no effect was found for protein content. A SNP in exon 17 (g.16101C>T) produces a non-conservative substitution of isoleucine to threonine at position 209 in the pre-protein (GenBank accession no. JN560175), but no significant associations with milk protein content were found. Finally, two SNPs located in exons 18 (g.16721C>A) and 19 (g.17826G>A) modify a putative target site for two bovine miRNA (bta-miR-631 and bta-miR-1224, respectively). Only the SNP in exon 18 showed a lightly significant additive genetic effect (PA) were consistent, as the expression of the CSN1S1 gene in animals with the CC genotype was approximately 1.8 times greater than the expression of the GG genotype. Further studies concerning cheese yield and casein content as well as functional studies of transcription factors and miRNA binding activity are needed to elucidate the function of these SNPs and their application to breeding schemes. © 2013 Elsevier B.V.