Substance use disorders are among the most common health problems of people involved with the criminal justice system. Scaling up addiction services in prisons is a global public health and human rights challenge, especially in poorly resourced countries. We systematically reviewed the prevalence of substance use in prison populations in low- and middle-income countries. We searched for studies reporting prevalence rates of nicotine, alcohol, illicit drug, and injection drug use during imprisonment in unselected samples of imprisoned people in low- and middle-income countries. Data meta-analysis was conducted and sources of heterogeneity were examined by meta-regression. Prevalence of nicotine use during imprisonment ranged from 5% to 87%, with a random-effects pooled estimate of 56% (95% confidence interval (CI): 45, 66) with significant geographical heterogeneity. Alcohol use varied from 1% to 76% (pooled prevalence, 16%, 95% CI: 9, 25). Approximately one-quarter of people (25%; 95% CI: 17, 33; range, 0–78) used illicit drugs during imprisonment. The prevalence of injection drug use varied from 0% to 26% (pooled estimate, 1.6%, 95% CI: 0.8, 3.0). Lifetime substance use was investigated in secondary analyses. The high prevalence of smoking in prison suggests that policies regarding smoking need careful review. Furthermore, the findings underscore the importance of timely, scalable, and available treatments for alcohol and illegal drug use by people involved with the criminal justice system.