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Abstract Background Substance use amongst university students is a recognized problem worldwide. Few studies have been carried out in this group in Botswana. These studies have been mostly limited to the use of alcohol and tobacco. Therefore, this study was designed to investigate the pattern of general substance use, its association with psychological distress and common socio-demographic factors among first-year undergraduates in a Botswana University. Methods A total of 401 students were interviewed using a modified W.H.O. student drug use questionnaire and the 12 item General Health Questionnaire (GHQ12) to assess the pattern of psychoactive substance use and its relationship with psychological distress amongst university students in Botswana. Results Alcohol was the most (31.9%) commonly used psychoactive substance. Age of debut for most psychoactive substances was between the ages of 15–18 years. Current use of alcohol (p = 0.045), amphetamine-type stimulants (p = 0.004) and benzodiazepines (p = 0.021) were associated with significant psychological distress. A positive relationship was observed between low participation in religious activities and substance use (OR = 4.63, 95%CI: 2.03–10.51), while a negative association was observed between not having a friend who uses drugs and substance use (OR = 0.44, 95%CI: 0.19–0.99). Conclusions There is a significant substance abuse problem in the undergraduate population in Botswana. Our findings followed the global trend, with alcohol being the most commonly used substance. Religious participation demonstrates potential to be one of the solutions to this problem, but how to harness its seemingly protective influences is a field for further study.