Pelvic masses present diagnostic challenges due to their complex origins and non-specific imaging characteristics. The accurate preoperative evaluation of pelvic masses plays a crucial role in guiding clinical decision making. This study sought to compare the efficacy and safety of ultrasound (US)-guided transluminal (transvaginal/transrectal) and transabdominal core needle biopsy (CNB) in the diagnosis of pelvic masses.A retrospective cohort study was conducted of 305 patients with pelvic masses, who were divided into the following two groups based on the biopsy approach: the US-guided transabdominal CNB group (the transabdominal group) and the transvaginal/transrectal CNB group (the transluminal group). The biopsy time, intraoperative blood loss, diagnostic accuracy, incidence of complications, and postoperative pain levels were evaluated and compared between the two groups.US-guided biopsy was successfully performed in 305 patients. There were no significant differences between the two groups in terms of accuracy (98.7% vs. 99.3%), biopsy time (98.2±28.7 vs. 94.5±27.7 s, P=0.25), number of puncture needles (2.8±0.7 vs. 2.7±0.8, P=0.61), blood loss (4.3±1.7 vs. 4.6±1.4 mL, P=0.21), or post-puncture visual analogue scale (VAS) score after puncture (2.8±1.2 vs. 2.9±1.2, P=0.52). The long and short diameters of the masses were greater in the transabdominal group (107.5±36.1 mm; 74.7±26.6 mm) than the transluminal group (65.6±37.6 mm; 45.0±23.0 mm; both P<0.001). The diagnostic ability of the transluminal (transvaginal/transrectal) CNB in deep pelvic lesions was better than that of the transabdominal CNB (P<0.001).US-guided transluminal (transvaginal/transrectal) and transabdominal CNB have comparable diagnostic efficacy and safety for pelvic masses. Transvaginal or transrectal biopsy has substantial clinical value in the evaluation of deep pelvic masses.Copyright © 2025 AME Publishing Company. All rights reserved.