Objective: To assess the association between genetic variants of transferrin receptor 2 (TFR2) exon 4 and anemia status and to describe the expression levels of several cytokines, hepcidin, soluble transferrin receptor and erythropoietin. Methods: Institutional based comparative study was done randomly to recruit 106 pregnant women who attended antenatal care in three different health centers in Boyolali Regency, Central Java from May 2015 to September 2015. DNA was extracted from peripheral blood samples of selected pregnant women and sequencing was done for TFR2 exon 4. Furthermore, enzyme-linked immunosorbent assay was conducted to measure the expression levels of interleukin 6, interleukin 4, transforming growth factor β and iron-metabolism related proteins such as hepcidin, soluble transferrin receptor, and erythropoietin. Gene alignment was performed by using a CLUSTAL W program. Collected data were analyzed statistically by using parametric and nonparametric tests with Statistical Product and Service Solutions (SPSS) 20.0 for Windows. Results: Three novel genetic variants from TFR2 exon 4 (position 603, 605 and 606) were associated with anemia status. Moreover, the expression levels of interleukin 6, interleukin 4, transforming growth factor β and erythropoietin were higher in anemic pregnant women than those of nonanemic pregnant women but only erythropoietin level reached statistical significance. These results were followed by decreases of hepcidin and soluble transferrin receptor levels. Conclusions: Various factors contribute to anemia prevalence among pregnant women in Boyoali Regency, Central Java, Indonesia. Our novel findings showed that TFR2 exon 4 has 3 mutational sites in position 603, 605 and 606. These novel genetic variants may provide a new insight into the role of TFR2 in anemia.