In the era of life-prolonging antiretroviral therapy, chronic fatigue is one of the most prevalent and disabling symptoms of people living with HIV/AIDS, yet its measurement remains challenging. No instruments have been developed specifically to describe HIV-related fatigue. We assessed the reliability and construct validity of the HIV-Related Fatigue Scale (HRFS), a 56-item self-report instrument developed through formative qualitative research and designed to measure the intensity and consequences of fatigue as well as the circumstances surrounding fatigue in people living with HIV. The HRFS has three main scales, which measure fatigue intensity, the responsiveness of fatigue to circumstances and fatigue-related impairment of functioning. The functioning scale can be further divided into subscales measuring impairment of activities of daily living, impairment of mental functioning and impairment of social functioning. Each scale demonstrated high internal consistency (Cronbach's alpha=0.93, 0.91 and 0.97 for the intensity, responsiveness and functioning scales, respectively). The HRFS scales also demonstrated satisfactory convergent validity when compared to other fatigue measures. HIV-Related Fatigue Scales were moderately correlated with quality of nighttime sleep (rho=0.46, 0.47 and 0.35) but showed only weak correlations with daytime sleepiness (rho=0.20, 0.33 and 0.18). The scales were also moderately correlated with general mental and physical health as measured by the SF-36 Health Survey (rho ranged from 0.30 to 0.68 across the 8 SF-36 subscales with most >0.40). The HRFS is a promising tool to help facilitate research on the prevalence, etiology and consequences of fatigue in people living with HIV.