Objectives The retroauricular fascia flap (RFF) is one of the most commonly used vascularized linings for auriculocephalic sulcus reconstruction in staged total auricular reconstruction. This study aims to investigate the histomorphometric features regarding the retroauricular fascia. Methods Histological evaluation included qualitative observation and quantitative analysis of sections of RFF stained with hematoxylin and eosin, Masson's trichrome, Elastica van Gieson, CD31, and Lyve-1. Ultrasonographic evaluation included measurement of the thickness of the superficial layer of the retroauricular fascia (RFF origin) at three different positions in microtia patients. P < 0.05 was considered statistically significant. Results RFF was a thin, highly organized layer with mainly collagen fibers. From its superior to inferior portions, the percentage of collagen fibers differed significantly (superior 87.57 +/- 10.85%, middle 68.29 +/- 29.02%, inferior 53.31 +/- 33.33%, p < 0.05). The percentages of elastic fibers in the superior (4.86 +/- 5.17%) and middle (5.05 +/- 5.37%) areas were higher than that in the inferior (2.14 +/- 2.42%, p < 0.05). RFF blood vessel density (20x magnification) decreased significantly from the superior to inferior portions (superior 6.39 +/- 1.18, middle 5.17 +/- 1.15, inferior 2.67 +/- 0.78, p < 0.05). Lymphatic vessel density (20x magnification) also decreased significantly from the superior to inferior regions (superior 6.80 +/- 0.62, middle 5.26 +/- 1.17, inferior 2.11 +/- 0.46, p < 0.05). Thickness of the superficial layer of retroauricular fascia increased significantly from the superior to inferior regions (superior 0.29 +/- 0.06 mm, middle 0.36 +/- 0.09 mm, inferior 0.53 +/- 0.14 mm, p < 0.001). Conclusions From cranial to caudal, the RFF became thicker, less elastic, and less vascularized, and contained fewer lymphatic vessels. Therefore, when the retroauricular fascia is large enough, the superior portion would be preferred for RFF in auriculocephalic sulcus reconstruction.