Female adnexal tumor of probable Wolffian origin (FATWO) is a rare gynecologic tumor. We describe a case of 53-year-old female patient in whom an adnexal mass was found. Microscopic examination revealed that the tumor arose in the adnexal soft tissue, composed of bland cells with an admixture of solid and sieve-like patterning, while presenting a high mitotic activity. Tumor cells were positive for Vimentin, CD10, and hormone receptors, while showing variable expression for sex cord-stromal markers, and was negative for GATA binding protein 3 (GATA-3), and thyroid transcription factor 1 (TTF1). The definitive diagnosis was FATWO. Subsequently, we conducted next-generation sequencing (NGS) in this case, and a CTNNB1 (c.98C>
G, p.S33C) mutation was detected. The patient underwent tumor resection, hysterectomy, and bilateral adnexectomy, followed by annual computed tomography scans for monitoring. No evidence of recurrence or metastasis was observed at the 2-year postoperative follow-up. To the best of our knowledge, this is the fourth study having performed NGS on a FATWO. To further elucidate this rare neoplasm and improve the accuracy of diagnosis, we conducted a comparative analysis of the clinicopathological, immunohistochemical, and molecular features of our case with those previously reported in the literature, subsequently discussing the differential diagnosis.