Objectives Women with undiagnosed pelvic lesions are often referred for evaluation and treatment. Transvaginal ultrasound-guided fine needle aspiration ( TVUS- FNA) biopsy can assist in making management decisions. We describe our experience with this modality. Methods We performed a retrospective chart review of all women who had a TVUS-FNA biopsy between January 2004 and December 2014. Charts were reviewed for clinicopathologic information. The pathologic results of the TVUS- FNA were compared with the final diagnosis. Results Fifty-nine women underwent TVUS- FNA; the median age was 66 years (range 27-85). Thirty-three lesions were evaluated by fine-needle aspiration biopsy of the solid structure and 26 by aspiration of fluid for cytology. Pathologic feasibility rate was 88% (52/59). Of those with evaluable tissue, the sensitivity of the procedure was 100% and the specificity 92%. Considering the seven inconclusive results, the procedure had sensitivity of 88% (29/33) and specificity of 88% (23/26). Overall accuracy of TVUS- FNA for this patient cohort was 85%. No patient characteristics were found to distinguish between accurate and inaccurate or inconclusive TVUS- FNA result. No complications were noted. Conclusions TVUS- FNA offers an excellent modality for the diagnosis and management of deep pelvic lesions otherwise not amenable for histologic evaluation.