Objective. This systematic review aims to synthesize articles, abstracts and reports of HIV prevalence studies conducted among men who have sex with men (MSM) and female sex workers (FSW) in Latin America and the Caribbean (LAC). Materials and methods. Authors searched online databases and collected gray literature on HIV prevalence among MSM and FSW from LAC. Year, location, sampling methodology, study design, sample size, HIV prevalence and confidence intervals were abstracted. Results. A total of 73 studies, dating from 1986 to 2010 were included. The median prevalences for MSM and FSW were 10.6% (interquartile range: 7.4-17.4) and 2.6% (IQR: 0.6-4.2), respectively. Variability was high, especially for MSM. The majority of studies recruited participants using convenience methods. Conclusion. HIV prevalence among MSM was higher than that among FSW. Sampling techniques should be standardized for future studies, prioritizing probability methods.