Background: Patterns of polydrug use among people who inject drugs (PWID) may be differentially associated with overdose as well as with unique risk factors for HIV transmission. This dissertation examines patterns of polydrug use among PWID in Tijuana, Baja California, Mexico, and San Diego, California and the relationship of these patterns to HIV risk behaviors and overdose. Methods: These analyses use data from two prospective cohort studies of PWID in San Diego (N=576) (Chapter 2) and Tijuana (N=735) (Chapters 2-4). Chapter 2 assesses prevalence and correlates of methamphetamine and heroin coinjection in a binational sample of PWID from both San Diego and Tijuana using logistic regression. Chapter 3 applies latent class analysis to identify classes of polydrug use and multinomial logistic regression to determine associations with HIV risk behaviors and overdose among PWID in Tijuana. Chapter 4 applies latent transition analysis to describe dynamic statuses of polydrug use at baseline and 6 month follow up, determine probabilities of transitioning between statuses, and examine whether these probabilities are modified by self-report needed for help for drug use. Results: Chapter 2 findings show that prevalence of heroin and methamphetamine co-injection in the past 6 months was 39.9% overall, was higher in Tijuana (55.8%) than in San Diego (19.8%), and was significantly associated with HIV injection risk behavior in both locations and associated with overdose only in San Diego. Chapter 3 findings show that PWID in Tijuana exhibited 5 distinct patterns of substance use behaviors. Compared to primarily heroin injectors, polydrug and polyroute class membership was associated with HIV injection and sexual risk behaviors. Chapter 4 findings showed that over a third (39.1%) of PWID in Tijuana transitioned to a different subgroup of heroin and methamphetamine polydrug use over 6 months. Those who reported greater need for help for drug use were more likely to transition to subgroups characterized by greater polydrug and polyroute use 6 months later. Conclusions: Findings highlight the heterogeneity in substance use patterns among PWID in Tijuana and San Diego and demonstrate that polydrug and polyroute users are a high-risk subgroup who may require more tailored prevention and treatment interventions.