This study focused on the use of antibiotics on small, medium and commercial-sized dairy farms in the central region of Zambia and its relationship to antibiotic resistance in Escherichia coli. A stratified random sample of 104 farms was studied, representing approximately 20% of all dairy farms in the region. On each farm, faecal samples were collected from a random sample of animals and a standardised questionnaire on the usage of antibiotics was completed. An E. coli isolate was obtained from 98.67% (371/376) of the sampled animals and tested for resistance to six classes of antibiotics. The estimated prevalence of resistance across the different farming systems was: tetracycline (10.61; 95%CI: 7.40-13.82), ampicillin (6.02; 95%CI: 3.31-8.73), sulfamethoxazole/ trimethoprim (4.49; 95%CI: 2.42-6.56), cefpodoxime (1.91; 95%CI: 0.46-3.36), gentamicin (0.89; 95%CI: 0.06-1.84) and ciprofloxacin (0%). Univariate analyses indicated certain diseases, exotic breeds, location, farm size and certain management practices as risk factors for detection of resistance, whereas multivariate analyses showed an association with lumpy skin disease and a protective effect for older animals (>25 months). This study has provided novel insights into the drivers of antibiotic use and their association with antibiotic resistance in an under-studied region of Southern Africa.