South Africa is a large country of approximately 1.22 million km2, made up of nine provinces with three climatic zones. Farming in the country is mostly defined by regional differences. Of the different organisms isolated from milk samples of dairy cows, Staphylococcus aureus poses a challenge to maintain udder health and wholesome dairy products for human consumption. Antibiotic resistant bacteria are therefore a potential health hazard. The objective of this study was to investigate the seasonal and regional relationships of antibiotic resistance of S. aureus, of which little is known. This study was undertaken to evaluate a data set of 3410 S. aureus isolates, taken from milk samples with somatic cell count > 400 000 cells/mL from commercial dairy herds. These isolates were tested for antimicrobial susceptibility using the Kirby Bauer method for ampicillin, cloxacillin, penicillin G, clindamycin, oxy-tetracycline, cephalexin, cefuroxime and tylosin. The samples were from 830 dairy herds, out of the estimated 2000 commercial dairy herds in South Africa. All the antibiotics tested, except for cephalosporins, showed a predicted prevalence of resistance of above 50% in most provinces, which is a concern. The lowest prevalence of resistance to the majority of the categories of antibiotics tested was present in KwaZulu-Natal during spring. The cephalosporins had the lowest levels of prevalence of bacterial resistance in Gauteng during winter. Resistance patterns of S. aureus to the eight antibiotics varied in the different seasons and provinces, possibly because of different weather conditions, and action and spectrum of antibiotics.