Simple Summary: Alpha-lactalbumin (α-LA) is a major whey protein component in mammalian milk, such as human (approximately 36%), bovine (approximately 17%), and other species, etc., It is involved in the regulation of lactose synthesis and has high nutritional value, especially in infant formula. Previous studies have confirmed that bovine α-LA gene 5 -flanking region has single nucleotide polymorphisms (SNPs), but little is known about polymorphisms in other regions, especially sequence coding for amino acids in protein (CDS) and their adjacent non-coding regions, including Chinese Holstein dairy cows. This study focused on investigated SNPs in the CDS and their adjacent non-coding regions of the α-LA gene in Chinese Holstein dairy cows, and assessed the association between SNPs and lactation traits. Sequence alignment showed that a potential SNPs (562th, G/A) in CDS2 region affect protein spatial structure, suggesting that this SNPs might affect the lactation traits of cows (milk type (Holstein and Jersey), and non-milk type (Bos Taurus)) need more in-depth study. More importantly, a novel SNPs at 1847th (T/C) bp in non-coding region near CDS4 was significantly associated with milk lactose composition, and lactose contents were significantly correlated with milk protein content, indicating that the SNPs could be used as a novel potential molecular marker for lactation traits in Chinese Holstein dairy cows.Abstract: Alpha-lactalbumin (α-LA) is a major whey protein in bovine and other mammalian milk, which regulates synthesis of lactose. Little is known about its genetic polymorphism and whether can be used as a potential marker for dairy ingredients, milk yield traits, and milk properties. To investigate its polymorphisms and their relationship with milk lactation traits in Chinese Holstein dairy cows, single-strand conformation polymorphism method (PCR-SSCP) and direct sequencing method were used to mark the α-LA gene SNPs. AA (0.7402) and AB (0.2598) genotypes were screened out by PCR-SSCP bands analysis in two independent populations. Direct sequencing revealed that there is one SNP at 1847th (T/C) bp in noncoding region of α-LA gene with highly polymorphic (0.5 < PIC = 0.5623 or 0.5822), of which T is in AA genotype while C in AB. Association analysis also showed that lactose content (p < 0.05) was negatively correlated with fat and protein contents within subgroup, indicating that the SNPs (1847th, T/C) in α-LA gene could be used as a novel potential molecular marker for lactation traits in Chinese Holstein dairy cows.