Following experts' consensus, waist circumference (WC) is the best anthropometric obesity index. However, different anatomic sites are used, and currently there is no universally accepted protocol for measurement of WC. In this study, we compare the associations between WC measured at different sites with total visceral adipose tissue (VAT) volume and cardiometabolic risk. Cross-sectional data were obtained from 294 adults and 234 children and adolescents. In addition, longitudinal data were provided in 75 overweight adults before and after dietary-induced weight loss. WC was measured below the lowest rib (WC(rib)), above the iliac crest (WC(iliac crest)), and midway between both sites (WC(middle)). Volumes of VAT and abdominal subcutaneous adipose tissue (SAT) were obtained using MRI. Cardiometabolic risk included blood pressure, plasma lipids, glucose, and homeostasis model (HOMA index). WC differed according to measurement site as WC(rib) < WC(middle) < WC(iliac crest) (P < 0.001) in children and women, and WC(rib) < WC(middle), WC(iliac crest) (P < 0.001) in men. Elevated WC differed by 10-20% in females and 6-10% in males, dependent on measurement site. In men and children, all WC had similar relations with VAT, SAT, and cardiometabolic risk factors. In women, WC(rib) correlated with weight loss-induced decreases in VAT (r = 0.35; P < 0.05). By contrast, WC(iliac crest) had the lowest associations with VAT and cardiometabolic risk factors in women. Each WC had a stronger correlation with SAT than with VAT, suggesting that WC is predominantly an index of abdominal subcutaneous fat. There is need for a unified measurement protocol.