The Uterine Artery Pulsatility Index (UAPI) is an ultrasound measure of tumour vascularity. In this study, we hypothesised that a UAPI ≤ 1 (high vascularity) would identify women with gestational trophoblastic neoplasia (GTN) at increased risk of resistance to first-line single-agent methotrexate (MTX-R). Single-centre cohort study. Charing Cross Hospital, a UK national centre for the treatment of trophoblastic disease. All women with a GTN FIGO score 5-6 treated with methotrexate (n = 92), between 1999 and 2011, at Charing Cross Hospital. UAPI was measured before the start of chemotherapy, and women were monitored for the development of MTX-R. Frequency of MTX-R in women with UAPI ≤ 1 compared with UAPI >1. UAPI was measured before chemotherapy in 73 of 92 women with GTN FIGO score 5-6. UAPI ≤ 1 predicted MTX-R independent of the FIGO score (hazard ratio 2.9, P = 0.04), with an absolute risk of MTX-R in women with a UAPI ≤ 1 of 67% (95% CI 53-79%) compared with 42% (95% CI 24-61%) with a UAPI >1 (P = 0.036). Our results suggest UAPI is an independent predictor of MTX-R in women with FIGO 5-6 GTN.