Study objectives To clinically validate the Flinders Fatigue Scale (FFS) as a brief measure of daytime fatigue, and to derive cut-off scores to classify fatigue severity. Method The FFS was administered to 439 adult volunteers from the general population, 292 adults with insomnia, 132 adults with Obstructive Sleep Apnoea (OSA) and 66 adults with Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME), together with the Fatigue Severity Scale (FSS) and the Epworth Sleepiness Scale (ESS). Results A factor analysis revealed a single factor solution for the seven-item scale (67% of total variance), although a better fit was obtained for a modified six-item version (75% of total variance). Group FFS scores varied in accordance with theorised fatigue levels, with CFS/ME and insomnia samples reporting significantly higher fatigue than OSA and volunteer samples. Good convergent validity was established with the FSS for volunteer (r = 0.67) and CFS/ME samples (r = 0.61). Excellent discriminant validity with the ESS was observed for the insomnia (r = −0.08) and CFS/ME groups (r = 0.03), while a small-to-moderate correlation was found within the volunteer sample (r = 0.29). Cut-off scores were identified to categorise borderline (13–15), moderate (16–20) and severe (≥21) fatigue. Conclusions The FFS is a reliable and valid instrument to quantify subjective daytime fatigue. Sensitivity and specificity analyses indicate scores that best discriminate insomniacs and CFS/ME populations from a non-clinical population. However, it is proposed that the data can also be used to indicate the severity of fatigue by reference to these first two groups.