Antibiotic Residues in Raw Cow Milk Collected from Smallholder Dairy Farms in Kasama and Mbala, Zambia

<b>Background/Objectives:</b> The deposition of antibiotic residues in animal source foods has become a global public health threat. This study aimed to assess antibiotic class residues in raw cow milk from smallholder dairy farms in Mbala and Kasama, Zambia. <b>Methods:</b> A cross-sectional study was conducted, in which 93 milk samples (54 from Mbala and 39 from Kasama) were randomly collected from lactating cows on 56 farms between May and June, 2025. The samples were analyzed using the Charm II assay for beta-lactams, tetracyclines, macrolides, sulfonamides, and aminoglycosides. A total of 100 mL of milk was collected in sterile plain tubes, placed in a cooler box with ice packs, and transported to the district laboratory's freezer and then delivered to the Central Veterinary Research Institute at (-18 to -20 °C), where they were stored at -20 °C. Statistical significance between districts was determined using Pearson's chi-square, and associations between a district and the occurrence of antibiotic residues in milk were evaluated using logistic regression. Data were analyzed using Stata 14.2 at a 95% confidence level (<i>p</i> = 0.05). <b>Results:</b> A total of 91.4% (<i>n</i> = 85) of samples had antibiotic residues above EU/MRLs, with mean positive samples being 0.91 ± 0.28 and a significant association between a district and residue occurrence (OR = 0.086; <i>p</i> = 0.025). Approximately 44.1% of the samples had multiple antibiotic residues. Approximately 82.1% of samples from Kasama and 98.1% from Mbala had antibiotic residues (<i>p</i> = 0.006). Approximately 68.8% of samples had sulphonamides, and 58.1% macrolides, indicating their widespread use. Tetracyclines were 12.9%, beta-lactams 9.7%, and aminoglycosides 2.2%. <b>Conclusions:</b> A majority of milk samples had antibiotic residues above EU/MRLs, raising public health threats and necessitating the development and implementation of policies.