Body condition of ungulates is a determinant of fecundity and survival rates. Ultrasonography and body condition scoring techniques allow reliable estimation of body fat but may not be feasible to employ in some circumstances. A reliable blood chemistry index for assessing relative condition of different ungulate populations or groups would be useful in ongoing population monitoring programs. We provided a nutrition supplement (treatment) to a group of free-ranging mule deer (Odocoileus hemionus) during 2 consecutive winters in southwest Colorado. In late February each year, we evaluated whether percent body fat and serum concentrations of total thyroxine (T4), total triiodothyronine (T3), free thyroxine (FT4), and free triiodothyronine (FT3) were higher among treatment deer than an adjacent group of deer that did not receive treatment (control). As a corroborative analysis, we modeled body fat as a function of thyroid hormone concentrations and morphometric variables. Estimated body fat of treatment deer averaged 12.3% (SE = 0.327), whereas estimated body fat of control deer averaged 7.0% (SE = 0.333) during the 2 winters of study. Concentrations of T4 and FT4 averaged 48.07 nanomole/L (SE = 3.80) and 12.61 picomole/L (SE = 1.04) higher, respectively, in treatment deer than control deer. Our optimal model of estimated body fat included T4, T4², FT4, and deer chest girth ( = −4.8015 − 0.0946 × T4 + 0.000603 × T4² + 0.1474 × FT4 + 0.1426 × chest girth, R² = 0.609). Serum thyroid hormones effectively discerned treatment deer from control deer and were related to estimated body fat. Ultrasound and body condition scoring should be used to estimate body fat whenever possible. However, in cases where only a blood sample can be obtained, we documented potential utility of T4 and FT4 during late winter for evaluating relative body condition of mule deer.