Background: It is estimated that between 34% and 50% of Australian women entering pregnancy are overweight and obese, which is associated with an increased risk in complications for both the woman and her infant. Current tools used in clinical and research practice for measuring body composition include body mass index (BMI), waist circumference and bioimpedance analysis. Not all of these measures are applicable for use during pregnancy due to a lack of differentiation between maternal and fetal contributions. While skinfold thickness measurement (SFTM) is increasingly being used in pregnancy, there is limited data and a lack of a standard tool for its use in overweight and obese pregnant women.Methods: We developed a standard tool for evaluating SFTM among women with a BMI ≥ 25 kg/m2. Forty-nine women were measured as part of a prospective cohort study nested within a multicentre randomised controlled trial (The LIMIT Randomised Controlled Trial). Two blinded observers each performed 2 skinfold measurements on the biceps, triceps and subscapular of each woman. Intraclass correlation coefficients (ICC) and standard error of measurement (SEM) were used to analyse SFTM, body fat percentage (BF%) and inter-observer variability.Results: The ICC for inter-observer variability in measurements were considered moderate for biceps SFTM (ICC = 0.56) and triceps SFTM (ICC = 0.51); good for subscapular SFTM (ICC = 0.71) and BF% (ICC = 0.74); and excellent for arm circumference (ICC = 0.97). The standard error of measurements ranged from 0.53 cm for arm circumference to 3.58 mm for the subscapular SFTM.Conclusion: Our findings indicate that arm circumference and biceps, triceps and subscapular SFTM can be reliably obtained from overweight and obese pregnant women to calculate BF%, using multiple observers, and can be used in a research setting.Trial registration: Australian and New Zealand Clinical Trials Registry ACTRN12607000161426.