α-Lactalbumin (α-LA) is one of the most abundant milk whey proteins among different mammalian species including domestic horse. The aim of this study was to screen for polymorphism in the 5’-flanking region of the equine α-LA (LALBA) gene and to assess the potential relationship of particular genotypes with LALBA gene expression variability (measured at the mRNA and protein levels) and with basic milk composition traits. Initial screening for LALBA gene 5’-flanking variants was conducted using direct sequencing of DNA derived from 96 horses representing 12 breeds (Polish Primitive Horse, Polish Coldblood Horse, Polish Warmblood Horse, Silesian, Hucul, Fiording, Haflinger, Shetland Pony, Welsh Pony, Arabian, Thoroughbred and Percheron). Association analysis of detected polymorphisms, gene expression and milk composition traits was carried out for 74 horses (Polish Primitive Horse, Polish Coldblood Horse and Polish Warmblood Horse breeds). Altogether 4 single nucleotide polymorphisms (SNPs) (c.−165G>C, c.−222A>G, c.−357C>A and c.−928C>T) were found in the LALBA gene 5’-flanking region (NC_009149.3 GenBank sequence, gene coordinates on ECA6: 67372475–67375877). Although bioinformatic prediction suggested that 3 of them may alter the consensus sequences for transcription factors, no significant associations between genotypes and LALBA gene expression were recorded. However, a significant relationship (P < 0.05) was noticed for c.−928C>T SNP genotypes and basic milk composition (fat and protein contents) of Polish Primitive Horse mares. Additionally, in our study the significant impact of horse breed and lactation period on LALBA gene expression and basic milk composition traits was revealed.