The aim of this study was to determine allele and phenotypic frequencies for single nucleotide polymorphism (SNP) of exon VI in the Pit-1 gene and their associations with type traits, milk yield, and days open. A total of 390 Holstein cows from Antioquia (Colombia) were genotyped for the Pit-1 Hinfi polymorphism by PCR-RFLP. We found AA, AB, and BB genotypes with a frequency of 0.03, 0.43, and 0.53, respectively. The A allele frequency was 0.35 and its presence in the genotype was associated with higher milk production, udder depth and body depth; the absence of the A allele in the genotype was associated with fewer days open. This study indicates that it is possible to develop breeding programs using the Pit-1 gene in Holstein cattle in the department of Antioquia.