Dual-energy X-ray absorptiometry (DXA) and isotope dilution technique have been used as reference methods to validate the estimates of body composition by simple field techniques; however, very few studies have compared these two methods. We compared the estimates of body composition by DXA and isotope dilution (¹⁸O) technique in apparently healthy Indian men and women (aged 19–70 years,n152, 48 % men) with a wide range of BMI (14–40 kg/m²). Isotopic enrichment was assessed by isotope ratio mass spectroscopy. The agreement between the estimates of body composition measured by the two techniques was assessed by the Bland–Altman method. The mean age and BMI were 37 (sd15) years and 23·3 (sd5·1) kg/m², respectively, for men and 37 (sd14) years and 24·1 (sd5·8) kg/m², respectively, for women. The estimates of fat-free mass were higher by about 7 (95 % CI 6, 9) %, those of fat mass were lower by about 21 (95 % CI − 18, − 23) %, and those of body fat percentage (BF%) were lower by about 7·4 (95 % CI − 8·2, − 6·6) % as obtained by DXA compared with the isotope dilution technique. The Bland–Altman analysis showed wide limits of agreement that indicated poor agreement between the methods. The bias in the estimates of BF% was higher at the lower values of BF%. Thus, the two commonly used reference methods showed substantial differences in the estimates of body composition with wide limits of agreement. As the estimates of body composition are method-dependent, the two methods cannot be used interchangeably.