Neck circumference (NC) has been proposed as a simple and practical tool, independently associated with cardiometabolic risk factors. However, the association of NC with inter-muscular adipose tissue (IMAT) is still to be determined. We aimed to examine the association of NC with thigh IMAT, and visceral adipose tissue (VAT) measured with computed tomography (CT) in overweight/obese women. 142 premenopausal overweight and obese Caucasian women participated in this cross-sectional study. NC was measured with an inextensible metallic tape above the thyroid cartilage according to International Society for Advancement of Kinanthropometry protocol. Thigh IMAT and VAT volumes were measured with a single cross-sectional CT. Regarding the covariates, fat mass (FM) was assessed with dual-energy x-ray absorptiometry and physical activity was objectively measured with accelerometry. NC was positively associated with thigh IMAT and VAT volumes (standardized β coefficient: β = 0.45, P-value = ≤0.001, β = 0.60, P = ≤ 0.001; respectively), which persisted after adjusting for age, height, overall FM or moderate-to-vigorous physical activity. Our findings show that NC is associated with thigh IMAT volume in overweight and obese premenopausal Caucasian women, regardless of the amount of lower-body fatness. These results suggest underscoring the relevance of NC as a marker of adipose tissue content in thigh skeletal muscle.