Microsatellite markers, also known as short tandem repeats (STRs), are important for marker-assisted selection to detect genetic polymorphism, and they are uniformly distributed in eukaryotic genomes. To analyze the relationship between microsatellite loci and lactation traits of Holstein cows in Xinjiang, 175 lactating cows with similar birth dates, the same parity, and similar calving dates were selected, and 10 STR loci closely linked to quantitative trait loci were used to analyze the correlation between each STR locus and four lactation traits (daily milk yield, milk fat percentage, milk protein percentage, and lactose percentage). All loci showed different degrees of genetic polymorphism. The average values of observed alleles, effective alleles, expected heterozygosity, observed heterozygosity, and polymorphic information content of the 10 STR loci were 10, 3.11, 0.62, 0.64, and 0.58, respectively. Chi-square and G-square tests showed that all populations of loci were in accordance with the Hardy–Weinberg equilibrium. Analysis of the correlation between STR locus genotype and lactation performance in the whole lactation period showed three loci (namely, BM143, BM415, and BP7) with no significant correlation with all lactation traits, two loci (BM302 and UWCA9) related to milk yield, three loci (BM103, BM302, and BM6425) related to milk fat percentage, two loci (BM302 and BM6425) related to milk protein percentage, and three loci (BM1443, BM302, and BMS1943) related to lactose percentage. The microsatellite loci selected in this study showed rich polymorphism in the experimental dairy cow population and were related to the lactation traits, which can be used for the evaluation of genetic resources and early breeding and improvement of Holstein dairy cows in Xinjiang.