The gene, vitellogenin (vtg) was cloned and characterized in the dojo loach (Misgurnus anguillicaudatus), an indigenous freshwater species in East Asia, in order to develop tools for detecting the effects of estrogenic endocrine-disrupting chemicals (EEDCs). Full-length cDNAs encoding seven distinct vtg transcripts (vtg1–7) were obtained. The corresponding deduced amino acid sequences (Vtg1–7) were divided into two types; type I (Vtg1–6; 89–99% identical), which contained both lipovitellin (Lv) and phosvitin (Pv), and type II (Vtg7), which contained Lv alone. Phylogenetic analysis revealed that the type I and type II Vtgs in the loach could be classified as VtgAo1 and VtgC types, respectively. Immuno-biochemical analyses using type-specific Vtg antisera revealed that VtgAo1 proteins appeared to be the major Vtg type in this species. Males were administered (aqueous exposure) either 17β-estradiol (E2) or 17α-ethinylestradiol (EE2), the results from which were used to determine that hepatic vtgAo1 expression was estrogen-sensitive. The precise classification of the loach vtg/Vtg products, as well as their induction profiles following the estrogenic stimulation, provide a basis for their use as sensitive biomarkers when EEDC activities are evaluated in the freshwater environments in East Asia.