HIV prevention programs targeting men who have sex with men, Blacks, and young adults commonly use measures of HIV knowledge as an important component of demonstrating overall program effectiveness. These scales, however, are rarely subjected to repeated analysis to confirm reliability and validity and the results of psychometric analysis rarely include subpopulation variations. In this study, we administered an adapted version of a previously validated HIV knowledge scale to participants of a large, city-wide HIV prevention program (n = 5,027) and performed psychometric analysis to determine if differences existed across populations. Analysis showed that the HIV knowledge scale performed poorly for men who have sex with men, but very well for transgenders. Results were similar for Blacks, Hispanics, and Whites, very poor for 30- to 39-year-olds, but very well for 60+ year olds. Findings underscore the need for further research on the measurement of HIV knowledge among high-risk populations and the importance of culturally appropriate survey items tailored to each population. © 2013 Society for Public Health Education.