We investigated the prevalence of folate deficiency and associated factors in a large population of Korean women of reproductive age during the COVID-19 pandemic. We utilized different cut-offs and evaluated age, year of testing, geographical region, and the utilization of serum homocysteine levels. Out of the 27,758 women evaluated, the overall prevalence of folate deficiency was 12.5% (<4 ng/mL, metabolic indicator) and 5.4% (<3 ng/mL, hematologic indicator). Homocysteine testing was observed in 8.4% of women, with 2.7% having elevated homocysteine levels (>15.4 µmol/L). According to our multiple logistic regression analysis, younger women, particularly those aged 20 to 24 years, tested in 2020, and from Jeolla province, Gyeongsang province, and Jeju Island, were identified as being more prone to folate deficiency. Receiver operating characteristic curve analysis demonstrated that a cut-off of >8.4 µmol/L provided the most accurate definition of folate deficiency with serum folate levels <4 ng/mL, while a cut-off of >8.8 µmol/L best defined folate deficiency with serum folate levels <3 ng/mL, with both cut-offs being lower than 15.4 µmol/L. Our study emphasizes the prevalence of folate deficiency, associated factors, and the role of homocysteine in planning nutritional support programs in Korea.