BACKGROUND: Low folate and vitamin B12 levels have negative effect on pregnancy outcomes but there is paucity of data on their levels among Indian women. Ferritin and haemoglobin are associated with maternal mortality and low birth-weight. Our aim was to estimate the prevalence of deficiency of serum folate and vitamin B12, and low levels of serum ferritin and blood haemoglobin among women of childbearing age from a rural population of South India. METHODS: We conducted a community-based cross-sectional study among 15-35 year women in a rural district. We used multistage stratified random sampling. Trained staff interviewed women to collect socio-demographic information and draw blood samples. We analysed samples for serum folate, vitamin B12, ferritin and blood haemoglobin levels and computed means and medians. We computed the proportion of deficiency based on cut-offs recommended by WHO. We examined the association of levels with age, parity and current pregnancy or breastfeeding by multi-variable regression using Stata 13.0. RESULTS: We recruited 979 women. One-fifth (185, 19%) were pregnant and one-fifth (196, 20%)were breastfeeding. Median serum folate levels were 2.5 ng/ml (IQR, 1.2-4.8), median vitamin B12 levels were 228.0 pg/ml (IQR, 121 - 390), median ferritin levels were 13.0 μg/l (IQR, 6.0 - 20.0) and median blood haemoglobin levels were 12.1 mg/dl (IQR, 10.7 - 13.6). Low levels of serum folate, vitamin B12, ferritin and haemoglobin were found in 57% (95% CI, 54-60%), 44% (95% CI, 41-48%), 46% (95% CI, 43-49%) and 28% (95% CI, 25-31%) respectively. Women with folic acid deficiency had two times higher prevalence of having vitamin B12 deficiency. In adjusted regression analysis folate levels were lower in older and breastfeeding women, but not associated with parity and were higher among pregnant women. Similar associations were not found with Vitamin B12 deficiency. Ferritin levels were higher in older women; but not associated with parity, pregnancy or breastfeeding. Haemoglobin levels were lower in pregnant and breastfeeding women. CONCLUSION: Our findings suggest that folic acid, vitamin B12 and iron deficiency are important public health problems in India. We observed that half of the women of childbearing age were deficient in these nutrients. Folic acid and vitamin B12 deficiencies co-exist and should be supplemented together.