Identifying clinical differences between opioid users (OU) and alcohol and other drug users (AOD) may help to tailor treatment to OU, particularly among the majority of OU who are not on opioid agonist treatments. Given the dearth of research on these differences, this study explored gender differences in demographic and clinical characteristics between OU and AOD. Participants (N = 506) were from a multisite, randomized controlled clinical trial of an Internet-delivered psychosocial intervention conducted in 2010-2011. Logistic regression models explored differences in demographic and clinical characteristics by substance use category within and between women and men. Women OU were more likely to be younger, White, employed, benzodiazepine users, and less likely to have children or use cocaine and cannabis than women AOD. Men OU, compared to men AOD, were more likely to be younger, White, younger at first abuse/dependence, benzodiazepine users, and reported greater psychological distress, but were less likely to be involved in criminal justice or use stimulants. Interactions by gender and substance use were also detected for age of first abuse/dependence, employment, and criminal justice involvement. These findings provide a nuanced understanding of gender differences within substance use groups to inform providers for OU seeking treatment.