Introduction and Aims. The increased availability of prescription opioids (PO) and non-medical prescription opioids (NMPO) has fundamentally altered drug markets and typical trajectories from initiation to high-risk use among people who use opioids (PWUO). This multi-site study explores trends in opioid initiation in three US cities and associations with sociodemographic factors, current drug use and overdose risk. Design and Methods. We analysed survey data from a cross-sectional study of PWUO in Baltimore, Maryland (n = 173), Boston, Massachusetts (n = 80) and Providence, Rhode Island (n = 75). Age of first exposure to PO, NMPO and heroin was used to calculate opioid of initiation, and multinomial regression was employed to explore correlates of initiating with each. Results. Thirty-three percent of PWUO initiated with heroin, 24% with PO, 18% with NMPO and 24% with multiple opioids in their first year of use. We observed a reduction in heroin initiation and gradual replacement with PO/NMPO over time. Women were more likely to initiate with NMPO [relative risk ratio (RRR) 2.4; 95% confidence interval (CI) 1.1, 5.0], PO (RRR 2.2, 95% CI 1.1, 4.4) or multiple opioids (RRR 2.1, 95% CI 1.1, 4.2), than heroin. PWUO initiating with NMPO had significantly higher current benzodiazepine use, relative to those initiating with heroin (RRR 3.2, 95% CI 1.4, 7.4), and a high prevalence of current fentanyl use (30%). Discussion and Conclusions. Our study highlights women and PWUO initiating with NMPO as key risk groups amid the changing landscape of opioid use and overdose, and discusses implications for targeted prevention and treatment. [Rouhani S, Park JN, Morales KB, Green TC, Sherman SG. Trends in opioid initiation among people who use opioids in three US cities. Drug Alcohol Rev 2020;39: [375][376][377][378][379][380][381][382][383]