This study aimed to investigate A1 and A2 polymorphisms of the beta-casein gene in crossbred Holstein-Zebu (HZ) cows from four herds in Northern Minas Gerais, Brazil. Blood samples were collected from 112 randomly selected cows, of wich 66 were at ½ HZ, and 46 were at ¾ HZ. DNA extraction was conducted using the phenol-chloroform method, and polymorphisms were evaluated using the amplification refractory mutation system - polymerase chain reaction technique. The results demonstrated that 72/112 (64.3%) cows were heterozygous (A1A2), 33/112 (29.5%) were homozygous for A2A2, and 7/112 (6.2%) were A1A1 homozygous. The percentage of A2A2 cows ranged from 10.7 to 41.4% among the evaluated herds. The frequencies of the A1 and A2 alleles were 38.4 and 61.6%, respectively. The A2 allele was the most frequent in both breeds. The ½ HZ cows were 1.8 times more likely (Odds Ratio) to belong to the A1A1 genotype. The distribution of genotypes was in Hardy-Weinberg equilibrium in three of the herds; however, the total of samples and the stratified samples according to breeds were not in equilibrium considering α = 0.05.