Background: Women of reproductive age (WRA) are a high-risk population for anemia and micronutrient deficiencies. Population-based biomarker data is needed to inform interventions and public health programs for anemia and birth defects prevention. Methods: Participants were 979 WRA (15-40y; not pregnant or lactating) from a population-based surveillance program in Southern India. Sociodemographic, nutritional, metabolic, and clinical data were collected using structured interviewers; a women’s health questionnaire (WHQ) was used to characterize reproductive histories and risk factors for birth defects. Venous whole blood samples were collected and analyzed for hemoglobin. Plasma, serum, and red blood cells (RBC) were processed and stored until batch analysis after data collection. RBC folate and serum folate (via WHO-recommended microbiologic assay), total vitamin B12 (via chemiluminescence), and anemia (via HemoCue 301 and automated Coulter HMX; AHA) were evaluated. Continuous variables were described using geometric means (GM) and 95% confidence intervals. Bayesian linear models were used to estimate the unadjusted and adjusted serum folate insufficiency threshold (sf-IT) for optimal neural tube defect prevention. Diagnostic accuracy parameters were calculated to evaluate screening methods for hemoglobin and anemia assessment using paired venous samples. Results: The prevalence of anemia (41.6%) and nutritional and metabolic risk factors for birth defects in this population was substantial. A total of 79.3% of women had RBC folate concentrations below the threshold for optimal NTD prevention. Among WRA with sufficient vitamin B12 status (≥221 pmol/L), the estimated sf-IT was consistent with previous Bayesian analyses. The estimated sf-IT increased in women with vitamin B12 deficiency (<148 pmol/L) or elevated MMA and was lower in WRA with elevated metabolic risk factors. The estimated prevalence of anemia was lower via portable hemoglobinometer compared to the AHA reference (36.3% vs. 41.6%), and its sensitivity was higher in WRA with iron deficiency and lower in WRA with elevated metabolic risk factors. Conclusions: The prevalence of risk factors for birth defects, including RBC folate insufficiency, and anemia were substantial in this population. Population-based biomarker surveillance in women of reproductive age is needed to inform anemia and birth defects prevention.