Background: Heroin production in Colombia has increased dramatically in recent decades, and some studies point to an increase in local heroin use since the mid-1990s. Despite this rapid increase, little is known about the effects of these activities on heroin injection within Colombia. One of the biggest concerns surrounding heroin injection is the potential spread of HIV through drug user networks. Objectives: This article examines injection risk behaviors among heroin injectors in the Colombian cities of Medellín and Pereira to explore the implications for possible increased HIV transmission within this group. Methods: A cross-sectional study used respondent-driving sampling to recruit a sample of 540 people who inject drugs (PWID) over 18 years of age (Medellín: n = 242, Pereira: n = 298). Structured interviews with each participant were conducted using the World Health Organization Drug Injection Study Phase II Survey. An HIV test was also administered. Results: Information regarding the socio-demographics, injection drug use, HIV risk and transmission behaviors, injection risk management, and HIV knowledge and prevalence of participants are reported. The study identified many young, newly initiated injectors who engage in risky injection practices. The study also found that HIV prevalence is fairly low among participants (2.7%). Conclusions/Importance: Findings indicate a potential risk for the spread of HIV among PWID in Colombia given their widespread sharing practices, high rate of new injector initiation, and unsafe syringe cleaning practices. Colombia has a possibly time-limited opportunity to prevent an HIV epidemic by implementing harm reduction interventions among young, newly initiated PWID.