Objective To study the anatomical location of retro-orbicularis oculi fat (ROOF) in the upper eyelid and to investigate how ROOF affects the appearance of the upper eyelid. Methods Twenty-eight Chinese hemifacial cadaver specimens were used (14 male cadavers; age range 52-82 years). In 28 hemifaces, the eyelids were dissected from the superficial to deep layers, and the appearance, location, extent, and surrounding tissue of ROOF were observed. Additionally, we observed the relationship between the upper eyelid morphology and ROOF of the upper eyelid in surgical patients who were treated in the plastic surgery department of Tongji Hospital affiliated with Huazhong University of Science and Technology in 2018. Results ROOF is a type of fascia adipose tissue that is located in a fat compartment between the muscles (the orbicularis oculi and frontalis muscles) and the orbital septum/frontalis fascia. In patients with hypertrophic ROOF, the upper eyelid appears as a heavy eyelid and as a drooping eyelid. And in patients with atrophic ROOF, the upper eyelid appears as a sunken eyelid. Conclusion ROOF is located in the fat compartment between the orbicularis muscle and the orbital septum/frontalis fascia. ROOF covers the entire upper eyelid and appears thinner medially and thicker laterally. It is continuous with the fat under the frontalis muscle and affects the appearance of the upper eyelid. It represents an important factor in upper eyelid surgery. No Level Assigned This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266..