High-dose-rate (HDR) interstitial brachytherapy is often included in standard-of-care for gynaecological cancers. Needles are currently inserted through a perineal template without any standard real-time imaging modality to assist needle guidance, causing physicians to rely on pre-operative imaging, clinical examination, and experience. While two-dimensional (2D) ultrasound (US) is sometimes used for real-time guidance, visualization of needle placement and depth is difficult and subject to variability and inaccuracy in 2D images. The close proximity to critical organs, in particular the rectum and bladder, can lead to serious complications. We have developed a three-dimensional (3D) transrectal US system and are investigating its use for intra-operative visualization of needle positions used in HDR gynaecological brachytherapy. As a proof-of-concept, four patients were imaged with post-insertion 3D US and x-ray CT. Using software developed in our laboratory, manual rigid registration of the two modalities was performed based on the perineal template’s vaginal cylinder. The needle tip and a second point along the needle path were identified for each needle visible in US. The difference between modalities in the needle trajectory and needle tip position was calculated for each identified needle. For the 60 needles placed, the mean trajectory difference was 3.23 ± 1.65° across the 53 visible needle paths and the mean difference in needle tip position was 3.89 ± 1.92 mm across the 48 visible needles tips. Based on the preliminary results, 3D transrectal US shows potential for the development of a 3D US-based needle guidance system for interstitial gynaecological brachytherapy.