Background and aims: Folate status is complex, complicated to assess and we lack consensus for laboratory determination.Total plasma homocysteine (Hcy) is a sensitive marker of folate status. The aim of this study was choosing a plasma total homocysteine (tHcy) measurement method and folate repletion level; assess the mutation C677T frequency for methylenetetrahydrofolate reductase (MTHFR) and the prevalence of hyperhomocysteinemia in healthy Majorcan women. Methods: The measurement methods were compared using 219 women. Folate status assessment was determined by plasma tHcy, serum and erythrocyte folate and C677T for MTHFR in 342 healthy women. The mutation frequency is established with 146 of them. Results: The measurement method comparison is summarized by Y = 1.013 (IC 95% 0.959, 1.069) X +0.829 (IC 95% 0.485, 1.170). The folate repletion level was set at Hcy ≤ 8.6 μmol/l. Women (67.1%) are heterozygotic and do not carry the mutation. Homozygotic frequency is 18.5%, significantly higher in women under 40 years ( P = 0.033). Hyperhomocysteinemia prevalence is 19.3%, and 51.7% in the younger group. Conclusions: Hcy determination by polarized fluorescence immunoassay reflects folate status. Levels ≤ 8.6 μmol/l suggest folate repletion, which is less frequent in homozygots. Population frequency for homozygotic-TT alleles for C677T–MTHFR reductase and hyperhomocysteinemia is significantly higher in women under 40 years.