We aimed to assess the effects of age, sex, body mass index (BMI), and anatomical site on skin thickness in children and adults with diabetes. We studied 103 otherwise healthy children and adolescents with type 1 diabetes aged 5-19 years, and 140 adults with type 1 and type 2 diabetes aged 20-85 years. The thicknesses of both the dermis and subcutis were assessed using ultrasound with a linear array transducer, on abdominal and thigh skin. There was an age-related thickening of both dermis (p<0.0001) and subcutis (p = 0.013) in children and adolescents. Girls displayed a substantial pubertal increase in subcutis of the thigh (+54%; p = 0.048) and abdomen (+68%; p = 0.009). Adults showed an age-related decrease in dermal (p = 0.021) and subcutis (p = 0.009) thicknesses. Pubertal girls had a thicker subcutis than pubertal boys in both thigh (16.7 vs 7.5 mm; p<0.0001) and abdomen (16.7 vs 8.8 mm; p<0.0001). Men had greater thigh dermal thickness than women (1.89 vs 1.65 mm; p = 0.003), while the subcutis was thicker in women in thigh (21.3 vs 17.9 mm; p = 0.012) and abdomen (17.7 vs 9.8 mm; p<0.0001). In boys, men, and women, both dermis and subcutis were thicker on the abdomen compared to thigh; in girls this was only so for dermal thickness. In both children and adults, the skin (dermis and subcutis) became steadily thicker with increasing BMI (p<0.0001). Skin thickness is affected by age, pubertal status, gender, BMI, and anatomical site. Such differences may be important when considering appropriate sites for dermal/subcutaneous injections and other transdermal delivery systems.