Malawi adopted the Option B+ strategy in 2011. Its success in reducing MTCT depends on coverage and timing of HIV testing. We assessed HIV status ascertainment and its predictors during pregnancy. HIV status ascertainment was 82.3% (95%-CI 80.2–85.9) in the pre-Option B+ period and 85.7% (95%-CI 83.4–88.0) in the Option B+ period. Higher HIV ascertainment was independently associated with higher age, attending ANC more than once, and registration in 2010. The observed high variability of HIV ascertainment between sites (50.6%–97.7%) and over time suggests that HIV test kits shortages and insufficient numbers of staff posed major barriers to reducing MTCT.