Abstract: Genetic variation of five microsatellite loci BM1818, BM143, BM1443, BM1905 and BM711 which were closely linked to milk composition traits was analyzed in 90 Hebei Holstein cows with nondenaturing polyacrylamide gel electrophoresis. Allele frequency, heterozygosity, polymorphic information content, the effective number of alleles of five microsatellite loci were calculated. Relationships between five microsatellite loci and milk composition traits in Hebei Holstein cows were preliminarily analyzed by least squares linear model. The results indicated that microsatellite BM1818 had significant effect on dry matter percentage; microsatellites BM143 and BM711 had significant effects on milk yield at 305 d, fat percentage, protein percentage and dry matter percentage; microsatellite BM1443 had significant effect on milk yield at 305 d; microsatellite BM1905 had significant effects on milk yield at 305 d and protein percentage. 102 bp/120 bp at BM143, 160 bp/160 bp at BM1443, 182 bp/195 bp at BM1905, 182 bp/182 bp at BM711 were the most favourable genotypes for milk yield at 305 d. 100 bp/112 bp at BM143, 173 bp/188 bp at BM711 were the most favourable genotypes for fat percentage. 120 bp/120 bp at BM143, 177 bp/189 bp at BM1905, 180 bp/188 bp at BM711 were the most favourable genotypes for protein percentage. 278 bp/282 bp at BM1818, 100 bp/112 bp at BM143, 173 bp/190 bp at BM711 were the most favourable genotypes for dry matter percentage.