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Background: genetic improvement in Colombia is limited by the lack of phenotypical and genealogical information. Additionally, molecular markers are important tools for studying the genetic structure of populations. Objective: to determine the structure of a population of Holstein cows in Antioquia, Colombia. Methods: a population of 1,800 animals distributed over 178 herds in 11 municipalities of Antioquia (Colombia) was genotyped using PCR-RFLP for gene polymorphisms of bovine growth hormone, kappa casein, prolactin, and BoLA DRB3.2. Population structure parameters, such as all Wright's F-statistics, were calculated and Hardy Weinberg equilibrium was determined. Analysis of molecular variance was carried out and Nei distances were estimated to determine population differentiation. Results: the total population was in Hardy Weinberg equilibrium for the four genes; however, kappa casein gene was in disequilibrium in some of the populations. Genetic structure of populations shows little genetic differentiation between them. Furthermore, a trend for outcrossing was found in most populations; this may be due to incorporation of imported semen into Colombia's dairy farms. The most genetically distant populations were in Marinilla and Rionegro municipalities due to their technological level and geographic location, which places them outside the scope of artificial insemination programs with foreign semen. Conclusions: the genetic similitude between populations of Holstein cows in Antioquia is due to their geographic proximity; therefore, their management of genetic conditions is very similar. [ABSTRACT FROM AUTHOR]