Importance: The risk of hematoma formation after rhytidectomy is gender associated and can lead to postoperative complications. The literature to help explain and elucidate the mechanism behind this gender-associated risk is poorly developed and requires further investigation. Objective: The objective of this study was to compare facial skin micro-vessel density of female and male-to-female (MTF) transgender patients undergoing rhytidectomy to better understand the mechanism of gender-correlated hematoma risk factors. The authors hypothesized that transgender patients would have higher micro-vessel density compared with female patients. Design, Setting, and Participants: This was a prospective histopathological analysis of pre- and post-auricular facial skin samples from patients undergoing primary rhytidectomy. Patient clinical data and skin samples were collected. Histopathological slides were prepared and stained with CD-31, a marker of vessel endothelium, followed by image analysis allowing for micro-vessel stained pixel counts and calculated pixel density comparisons at a single academic hospital. Female, MTF transgender, and male patients >18 years of age were studied. Exposure: Patients undergoing primary rhytidectomy between 2015 and 2018. Main Outcomes: Gender-associated pre- and post-auricular micro-vessel pixel density. Results: Forty-one patients contributed skin samples for analysis. Post-auricular micro-vessel pixel density was greater than pre-auricular density (mean difference post-pre 0.359 percentage points [p.p.], standard error [SE] = 0.135, p = 0.009). The mean post-auricular micro-vessel pixel density was 1.60% (SE = 0.13 p.p.), 2.16% (SE = 0.19 p.p.), and 2.77% (SE = 0.34 p.p.) for female, transgender, and male patients, respectively (p = 0.016). Pre-auricular micro-vessel pixel density showed no difference among females, males, and transgender patients (p = 0.30). Gender was a strong predictor of increased post-auricular micro-vessel pixel density on stepwise linear regression, but it did not predict pre-auricular micro-vessel density. Both preoperative hair removal and a history of hypertension were associated with increased pre-auricular micro-vessel density. Conclusions and Relevance: Facial skin micro-vessel density differs by gender, in addition to pre- and post-auricular locations in patients undergoing rhytidectomy.