To determine the genetic variability and population structure of the β-lactoglobulin (LGB) locus in the Argentinian Holstein breed, milk samples from 12 herds at four locations in Córdoba, Argentina, were analysed. Allelic and genotypic frequencies of LGB variants were compared by contingency tables. A G-test was applied to detect Hardy–Weinberg equilibrium. LGB A allele frequency was 0·43 and B allele frequency was 0·57. Allelic and genotypic frequencies indicated great homogeneity among populations. Most of the populations were in Hardy–Weinberg equilibrium. According to the fixation index, populations were panmictics. The partition of the F-statistic demonstrated that mating was at random, although there was a slight excess of heterozygotes, and there were no genetic differences among populations. The Shannon–Weaver index showed that more than 96% of the total genetic diversity for this locus is attributable to the differences within each herd. The diversity among herds within locations and among locations accounted for <4% of the total variation. Phenograms obtained by the Nei procedure for genetic distances disclosed the separation of the herds into two groups, one with an average frequency of LGB B of 0·61 and the other with an LGB B frequency of 0·53. Locations defined two clusters, but the divergence among populations was not important. These results indicated that the Argentinian Holstein breed from Córdoba constitutes one population.