Background: Perianal fistula surgery can damage the anal sphincters which may cause faecal incontinence. By measuring regional pressures, 3D-HRAM potentially provides better guidance for surgical strategy in patients with perianal fistulas. The aim was to measure regional anal pressures with 3D-HRAM and to compare these with 3D-EUS findings in patients with perianal fistulas. Methods: Consecutive patients with active perianal fistulas who underwent both 3D-EUS and 3D-HRAM at a clinic specialised in proctology were included. A group of 30 patients without fistulas served as controls. Data regarding demographics, complaints, previous perianal surgical procedures and obstetric history were collected. The mean and regional anal pressures were measured with 3D-HRAM. Fistula tract areas detected with 3D-EUS were analysed with 3D-HRAM by visual coding and the regional pressures of the corresponding and surrounding area of the fistula tract areas were measured. The study was granted by the VUmc Medical Ethical Committee. Results: Forty patients (21 males, mean age 47) were included. Four patients had a primary fistula, 19 were previously treated with a seton/abscess drainage and 17 had a recurrence after previously performed fistula surgery. On 3D-HRAM, 24 (60%) fistula tract areas were good and 8 (20%) moderately visible. All but 7 (18%) patients had normal mean resting pressures. The mean resting pressure of the fistula tract area was significantly lower compared to the surrounding area (47 vs. 76 mmHg; p < 0.0001). Only 2 (5%) patients had a regional mean resting pressure < 10 mmHg of the fistula tract area. Using a Δ mean resting pressure ≥ 30 mmHg difference between fistula tract area and non-fistula tract area as alternative cut-off, 21 (53%) patients were identified. In 6 patients 3D-HRAM was repeated after surgery: a local pressure drop was detected in one patient after fistulotomy with increased complaints of faecal incontinence. Conclusions: Profound local anal pressure drops are found in the fistula tract areas in patients normal mean resting pressures. Fistulotomy may affect local sphincter pressure. This might influence surgical decision making in future.