Villin1 (VIL1) has a role in regulating actin dynamics, cell morphology, anti-apoptotic mechanisms, and epithelial-to-mesenchymal transition. Previously we reported VIL1 as a novel diagnostic marker for cervical adenocarcinoma (AC) with poor radioresponse. This study further investigated the diagnostic role of VIL1 in gynecological tumors especially endometrial AC. We recruited 107 patients with AC (41 tumors in the corpus and 66 tumors in cervix), most of whom treated by total abdominal hysterectomy. Immunohistochemical analysis revealed VIL1-positive tumors in 37% of cases; 10 of 41 corpus tumors and 30 of 66 tumors in the cervix. VIL1-positive tumors were further examined histologically and immunostained for epithelial cell surface marker, EpCAM, and mesenchymal stem cell marker, CD44. Most of these tumors were CD44 negative and EpCAM positive, and the cytoplasmic VIL1 immunoreactivity in endometrial AC was more selective than EpCAM in reflecting histological aggressiveness with high grade nuclear atypia. This study confirmed our previous finding of VIL1 as a diagnostic marker of cervical AC. In addition, VIL1 immunostaining was detected in 25% of endometrial AC cases. These results suggested the existence of an aggressive and VIL1-positive subtype of gynecological tumor.