Question How do rates of self-reported mistreatment and sources of mistreatment vary between lesbian, gay, bisexual, transgender, queer, and other sexual and gender minority (LGBTQ+) general surgery residents and their non-LGBTQ+ peers? Findings In this survey study of 6381 surgical residents, LGBTQ+ general surgery residents reported higher rates of discrimination, harassment, and bullying than their non-LGBTQ+ peers, with attending surgeons as the most common source. Despite reporting similar career satisfaction, LGBTQ+ residents were twice as likely to consider leaving their program and/or have thoughts of suicide. Meaning Mistreatment is a common experience for LGBTQ+ general surgery residents, and multifaceted interventions are necessary to develop safer and more inclusive learning environments. Importance Previous studies have shown high rates of mistreatment among US general surgery residents, leading to poor well-being. Lesbian, gay, bisexual, transgender, queer, and other sexual and gender minority (LGBTQ+) residents represent a high-risk group for mistreatment; however, their experience in general surgery programs is largely unexplored. Objective To determine the national prevalence of mistreatment and poor well-being for LGBTQ+ surgery residents compared with their non-LGBTQ+ peers. Design, Setting, and Participants A voluntary, anonymous survey adapting validated survey instruments was administered to all clinically active general surgery residents training in Accreditation Council for Graduate Medical Education-accredited general surgery programs following the 2019 American Board of Surgery In-Training Examination. Main Outcomes and Measures Self-reported mistreatment, sources of mistreatment, perceptions of learning environment, career satisfaction, burnout, thoughts of attrition, and suicidality. The associations between LGBTQ+ status and (1) mistreatment, (2) burnout, (3) thoughts of attrition, and (4) suicidality were examined using multivariable regression models, accounting for interactions between gender and LGBTQ+ identity. Results A total of 6956 clinically active residents completed the survey (85.6% response rate). Of 6381 respondents included in this analysis, 305 respondents (4.8%) identified as LGBTQ+ and 6076 (95.2%) as non-LGBTQ+. Discrimination was reported among 161 LGBTQ+ respondents (59.2%) vs 2187 non-LGBTQ+ respondents (42.3%; P < .001); sexual harassment, 131 (47.5%) vs 1551 (29.3%; P < .001); and bullying, 220 (74.8%) vs 3730 (66.9%; P = .005); attending surgeons were the most common overall source. Compared with non-LGBTQ+ men, LGBTQ+ residents were more likely to report discrimination (men: odds ratio [OR], 2.57; 95% CI, 1.78-3.72; women: OR, 25.30; 95% CI, 16.51-38.79), sexual harassment (men: OR, 2.04; 95% CI, 1.39-2.99; women: OR, 5.72; 95% CI, 4.09-8.01), and bullying (men: OR, 1.51; 95% CI, 1.07-2.12; women: OR, 2.00; 95% CI, 1.37-2.91). LGBTQ+ residents reported similar perceptions of the learning environment, career satisfaction, and burnout (OR, 1.22; 95% CI, 0.97-1.52) but had more frequent considerations of leaving their program (OR, 2.04; 95% CI, 1.52-2.74) and suicide (OR, 1.95; 95% CI, 1.26-3.04). This increased risk of suicidality was eliminated after adjusting for mistreatment (OR, 1.47; 95% CI, 0.90-2.39). Conclusions and Relevance Mistreatment is a common experience for LGBTQ+ surgery residents, with attending surgeons being the most common overall source. Increased suicidality among LGBTQ+ surgery residents is associated with this mistreatment. Multifaceted interventions are necessary to develop safer and more inclusive learning environments. This survey study explores the national prevalence of mistreatment and poor well-being for lesbian, gay, bisexual, transgender, queer, and other sexual and gender minority (LGBTQ+) surgery residents compared with their non-LGBTQ+ peers.