Hiperthyroid is execcesive thyroid function which increased thyroid hormone that suppressed TSH. Major etiologies of hyperthyroidism (60%-80%) caused by Graves disease. Graves disease occurs in up to 2% women 20-40 years old. Some of the genes implicated in this pathogenesis may encode thyroid stimulating hormones receptors (TSHR). The TSHR gene is located on chromosome 14q31, an area in which a Graves disease susceptibility locus (GD-1) has been mapped. The GD-1 locus is specifically linked to Graves’ disease. TSHR polymorphisms, resulting in amino acid substitutions, have been identified. Two of these are located in the extracellular domain of the receptor (Asp36His and Pro52Thr), and one is located in the intracellular domain (Asp727Glu). A germline mutation of TSHR codon 727 has been reported to be associated with Graves disease. The aim of this research is to analyze the polymorphism of codon D727E on hiperthyroid patient. Sixteen respondents with suspect hyperthyroidism were chosen. The blood were collected for TSH, FreeT4, Thyroglobulin Antibody and Thyroidperoxidase Antibody and DNA isolation. The polymorphic region of the target genes (gene TSHR codon 727) were amplified by polymerase chain reaction (PCR). PCR-Sequencing was used for polymorphism on codon D727E confirmation. The result showed there is no Polymorphism in gen TSHR codon D727E. The codon D727E variant of TSHR gene is not associated with Hyperthyroid in Sukoharjo population. The possibility that mutation may occur on another genes need to be further analyzed.