Objective:.Anaemia is associated with an increased risk of disease progression and all-cause mortality among HIV-infected individuals, regardless of the type of anaemia, but the magnitude of the risk is greater with more severe anaemia. Although anaemia PLWH has been extensively studied, the focus has primarily been on its prevalence and association with disease progression in untreated or poorly controlled HIV cases. This study aimed to investigate the prevalence, and factors associated with moderate-to-severe anaemia among virally suppressed HIV patients at a tertiary hospital in Zambia. Methods: We conducted a cross-sectional study of ART treated PLWH for at least 6 months between October 2023 and February 2024 at Livingstone University Teaching Hospital (LUTH). Sociodemographic, clinical, and laboratory were the data collected. Anaemia was defined based on the World Health Organisation (WHO) classification, as haemoglobin concentration lower than normal i.e. <12 g/dl in females and <13 g/dl in males. The primary outcome was moderate-to-severe anaemia defined as haemoglobin level below 10.9 g/dl based World Health Organisation (WHO) classification. Logistic regression was performed to identify factors associated with moderate-to-severe anaemia. Results: Among 823 participants with viral suppression, the overall prevalence of anaemia and moderate-to-severe anaemia was 29.4% (n=242; 95% confidence interval (CI): 26.3-32.6) and 14.2% (n=117, 95% CI: 11.7-18), respectively. Females exhibited significantly higher odds of moderate-to-severe anaemia compared to males (adjusted odds ratio (AOR) 2.65; 95% confidence interval (CI): 1.21-5.81). Lower lymphocyte count (AOR 0.542; 95% CI: 0.33- 0.90), higher BMI (AOR 1.06; 95% CI: 1.01-1.12), and Microcytosis (AOR 14.35; 95% CI: 7.33 - 28.08) were associated with increased odds of anaemia. Participants on non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimens had a higher likelihood of anaemia compared to those on integrase strand transfer inhibitor (INSTI) regimens (AOR 5.16; 95% CI: 1.02- 26.24). Conclusion: We found a higher prevalence of anaemia and moderate-to-severe anaemia in virally suppressed PLWH, suggesting factors beyond HIV contribute to the persistence of anaemia in this cohort. Female gender, lower lymphocyte count, higher BMI, low mean corpuscular volume, and NNRTI-based ART regimens were independently associated with moderate-to-severe anaemia. Further research is warranted to explain the underlying mechanisms and optimize clinical management to improve outcomes among virally suppressed PLWH.