Abstract Background Female sex workers (FSW) are at increased risk for HIV and other STI due to occupation-related risks and exposures. Long-distance truck drivers have been implicated in the spread of HIV, but less is known about HIV/STI risks of FSW servicing truck drivers, especially in North America. As part of an international collaborative pilot study, we interviewed FSWs servicing truck driver clients along two major transportation corridors to explore factors associated with recent STI symptoms. Methods A cross-sectional study of 200 FSW was conducted in Mexico: 100 from Nuevo Laredo (U.S. border); 100 from Ciudad Hidalgo (Guatemalan border). Eligibility criteria included age ≥18 years, speaking English or Spanish, and having ≥1 truck driver client in the past month. The main outcome was reporting any recent STI symptoms, defined as experiencing genital/anal warts, genital ulcers/sores, genital itching, or abnormal vaginal discharge in the past 6 months. Logistic regression was used to identify correlates of recent STI symptoms. Results Median age of FSW was 29 years, 74% were single, 87% had <9th grade education, and median income was 4000 pesos/month ($300 USD). Sex work occurred at a bar/cantina for 70%. One-quarter had never been tested for HIV, 53% reported lifetime drug use, 22% reported drinking alcohol before/during transactional sex and 17% reported recent STI symptoms. After controlling for age and study site, factors associated with STI symptoms were lifetime drug use (AOR 2.9, 95% CI 1.2-6.9), drug use before/during sex (AOR 2.8, 95% CI 1.1-7.1), alcohol use before/during sex (AOR 5.2, 95% CI 2.2, 12.6), forced sex ever (AOR 2.6, 95% CI 1.1-6.1), lifetime history of arrest (AOR 2.3, 95% CI 1.0-5.0), and being surveyed in Nuevo Laredo rather than Ciudad Hidalgo (AOR 4.8, 95% CI 2.0-10.0). Conclusions The associations we observed between recent STI symptoms and drug and alcohol use suggest that interventions are needed that promote consistent and effective safer sex practices, especially while under the influence of alcohol or other substances.