OBJECTIVE: Female and male surgical residents are treated differently based on their gender. The use of assessment tools can help obtain objectivity in surgical skills assessment, avoid gender bias, and promote equal learning opportunities. The objective of the study was to explore whether knowledge of gender causes bias in the rating of surgical skills, and whether the raters’ gender, surgical specialty, or experience affect ratings. DESIGN: The study is designed as a cross-sectional study, where an anonymous video showing surgical performance was rated by surgeons from different surgical specialties and different levels of surgical experience. The same video was presented to two randomized groups as either a male or female surgical trainee performing the procedure. SETTING: The participants used Objective Structured Assessment of Technical Skills (OSATS) to rate a video-recorded exam from a surgical skills course at Copenhagen Academy for Medical Education and Simulation (CAMES). The video rating was done through an internet-based platform and could thus be performed at home or at another location of choice. PARTICIPANTS: Novice and experienced surgeons from departments of gastrointestinal surgery, gynaecology, and urology in Denmark. RESULTS: There was no difference in OSATS rating score of the perceived female and male trainee (female trainee (n = 50):12.26 (SD = 3.08), male trainee (n = 57): 12.00 (SD = 3.28), p-value = 0.67). Rater characteristics: gender, age, surgical experience, and surgical specialty did not affect ratings. CONCLUSIONS: When OSATS a systematic assessment tool was used in rating surgical trainees we did not find any significant implicit gender bias. The rating was unaffected by the raters’ gender, age, surgical specialty, or experience.