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Anemia is an important public health challenge and accurate prevalence estimates are needed for program planning and tracking progress. While venous blood assessed by automated hematology analyzers is considered gold standard, most population‐based surveys use point‐of‐care diagnostics and capillary blood to estimate population prevalence of anemia. Several factors influence hemoglobin (Hb) concentration, including human and analytic error, analysis method, and type of instrument, but it is unclear whether biological variability exists between venous and capillary blood. The objective of this paper was to systematically review sources of Hb variability and the potential biological basis for venous and capillary differences. We use data from a recent survey in the state of Uttar Pradesh, India, to illustrate the implications on anemia prevalence estimates. Significant differences in Hb concentration between capillary and venous blood samples are common. Most but not all find capillary Hb concentration to be higher than venous. Instrument/method variability and human error play an important role, but cannot fully explain these differences. A normative guide to data collection, analysis, and anemia diagnosis is needed to ensure consistent and appropriate interpretation. Further research is needed to fully understand the biological implications of venous and capillary Hb variability. [ABSTRACT FROM AUTHOR]