The objective of the present study was to determine differences in predicting total and regional adiposity using the waist:height ratio (WHtR) calculated using different ‘waist’ measurements. Body composition of ninety-five males and 121 female Australian adults (aged 20 years and above) was measured using dual-energy X-ray absorptiometry. The WHtR was calculated using: (1) the narrowest point between the lower costal border and the top of the iliac crest (WHtR-W), and (2) at the level of the umbilicus (WHtR-A). Relationships between calculated WHtR and measured body composition, such as percentage body fat (%BF) and percentage trunk fat (%TF) were determined. Values obtained from WHtR-A were significantly greater than WHtR-W in both groups (P < 0·05). While no correlation differences between WHtR-W and WHtR-A in relation to body composition variables were observed, females showed significantly lower correlation with lean mass compared with BMI. Regression analyses showed that neither WHtR had an age influence on %TF estimation. Estimated %BF and %TF were comparable for both WHtR and also with estimated values using a BMI of 25 kg/m2. Sensitivity of excess %BF and %TF increased by using WHtR-A, particularly in females. In conclusion, the umbilicus measurement may be better than using the narrowest site in the WHtR calculation, particularly in females. To improve the screening ability of the WHtR and make comparisons between studies easier there may be a need to standardise the measurement location. Further studies are recommended to confirm the findings across different ethnic groups.