Introduction: Anemia is a global public health concern affecting mainly women of reproductive age (WRA) and preschoolers (PSC,6-59 months) due to their higher demand for iron. The etiology of anemia is multifactorial, and nutritional anemia is the most common type worldwide and is predominantly due to deficiencies in iron, vitamin B12, and folate. This study aimed to assess the associated factors, etiology, and prevalence of anemia among WRA and preschoolers in the GBAO region in Tajikistan. Methods: We conducted a cross-sectional study in the GBAO province of Tajikistan between April 2021 and September 2021, enrolling 500 WRA and 500 children aged 6-59 months from six districts of the GBAO region. The survey was administered through a structured questionnaire at the household level, and information was collected on sociodemographic characteristics, reproductive history, dietary intake, nutritional status, and maternal factors. Blood and stool samples were also collected for micronutrient deficiencies and helminthic infections. Data analysis began with univariate analysis, followed by multivariate logistic regression in Stata (version 18). Results: Biochemical assessment of 473 WRA and 390 preschoolers was conducted; 17.3% of WRA and 15.4% of PSC were anemic. After adjustment, the following factors were positively associated with anemia in WRA living in the GBAO region; body mass index ( overweight (OR 0.313, p value 0.020), gravidity (OR 0.326,p value 0.009), low ferritin (OR 8.549,p value <0.001), elevated serum transferrin receptor levels (OR 4.817,p value <0.001) and Vitamin B12 deficiency (OR 0.18,p value 0.050). In contrast, in children, maternal education (OR 2.35, p value 0.029), maternal anemia (OR 4.998, p value 0.001), age under 2 years (OR 2.6, p value 0.016), and low ferritin (OR 5.67, p value <0.001) showed a positive association with anemia. Conclusions: Anemia in women was strongly associated with low ferritin, high STFR, gravidity, BMI, and vitamin B12; in children, with maternal education, maternal anemia, age <24 months, and low ferritin. These findings underscore the urgent need for locally tailored interventions, both in the short term and the long term.