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Antimicrobial resistance (AMR) is a global public health problem affecting animal and human medicine. Poultry production is among the primary sources of income for many Zambians. However, the increased demand for poultry products has led to a subsequent increase in antimicrobial use. This study assessed the awareness of AMR and associated factors among layer poultry farmers in Zambia. A cross-sectional study was conducted among 77 participants from September 2020 to April 2021. Data was analysed using Stata version 16.1. The overall awareness of AMR among the farmers was 47% (n = 36). The usage of antibiotics in layer poultry production was high at 86% (n = 66). Most antibiotics were accessed from agrovets (31%, n = 24) and pharmacies (21%, n = 16) without prescriptions. Commercial farmers were more likely to be aware of AMR compared to medium-scale farmers (OR = 14.07, 95% CI: 2.09–94.70), as were farmers who used prescriptions to access antibiotics compared to those who did not (OR = 99.66, 95% CI: 7.14–1391.65), and farmers who did not treat market-ready birds with antibiotics compared to those who did (OR = 41.92, 95% CI: 1.26–1396.36). The awareness of AMR among some layer farmers was low. Therefore, policies that promote the rational use of antibiotics need to be implemented together with heightened surveillance activities aimed at curbing AMR.