OBJECTIVE The aims of this analysis were to determine if there is an association between serum levels of testosterone and estradiol with meibomian gland (MG) morphology and lipid layer thickness. METHODS The data used for this analysis were collected from postmenopausal women with and without dry eye disease. Meibography was used to assess MG dropout on the central two-thirds of the eyelid and biomicroscopy was used for assessing MG expressibility and meibum quality. Venous blood samples were drawn for serum hormone level analysis. The Kruskal-Wallis test and Spearman correlations were used for statistical analysis. RESULTS One hundred ninety-eight postmenopausal women with an average age of 61.2 (± 9.1) years were included in this analysis. Testosterone levels showed significant differences between MG dropout grades 1 and 4 (P = 0.002) and grades 2 and 4 (P = 0.01), whereas estradiol levels were different based on MG dropout (P = 0.53). No significant correlations were found between testosterone (r = 0.10, P = 0.17) or estradiol (r = 0.05, P = 0.45) and lipid layer thickness. CONCLUSIONS Testosterone levels were increased with MG dropout, which was significant between the mild and severe dropout groups, whereas no significant differences were found with estradiol and any MG assessment. Although the literature suggests an association of serum hormone levels and pathogenesis of dry eye disease in postmenopausal women, analysis of active sex steroid precursors and local tissue hormone levels may prove more useful.