Although primary prevention of HAV and HBV can be achieved through vaccination, the burden of HCV can only be reduced through behavioral interventions to reduce its risk factors. This study evaluated knowledge regarding transmission, clinical manifestations and prevention of viral hepatitis in Puerto Rico. We assessed the level of knowledge about HAV (six questions), HBV (12 questions) and HCV (eight questions) among non-institutionalized Puerto Rican adults aged 21-64 years. Demographic characteristics and self-reported knowledge of these infections were determined through a face-to-face interview. A mean knowledge score was computed by summing correct responses to each scale. Mean knowledge scores according to demographics were compared using ANOVA or the Kruskal-Wallis test. Mean knowledge scores for HAV, HBV and HCV infections were 2.6 ± 1.5, 6.1 ± 2.4, and 3.6 ± 1.1, respectively. For HAV and HBV infections, the mean knowledge score significantly (P < 0.05) increased with age, level of counseling received and number of sources of information. However, for HCV infection the mean knowledge score significantly increased with decreasing age, increased educational level and increased annual family income. Contrary to HBV, a higher HAV and HCV knowledge score was observed among individuals with history of vaccination for HAV and HBV, seropositive status for HAV and HCV, and history of drug use. A sizeable proportion of adults in this study demonstrated an inadequate level of knowledge, especially about transmission routes. Health education must be focused on transmission and prevention methods, including the availability of a vaccine for HAV and HBV, especially among those with chronic liver disease.