The COVID-19 pandemic has placed increased strain on health care workers and disrupted childcare and schooling arrangements in unprecedented ways. As substantial gender inequalities existed in medicine before the pandemic, physician mothers may be at particular risk for adverse professional and psychological consequences. To assess gender differences in work-family factors and mental health among physician parents during the COVID-19 pandemic. This prospective cohort study included 276 US physicians enrolled in the Intern Health Study since their first year of residency training. Physicians who had participated in the primary study as interns during the 2007 to 2008 and 2008 to 2009 academic years and opted into a secondary longitudinal follow-up study were invited to complete an online survey in August 2018 and August 2020. Work-family experience included 3 single-item questions and the Work and Family Conflict Scale, and mental health symptoms included the Patient Health Questionnaire-9 (PHQ-9) and Generalized Anxiety Disorder-7 scale. The primary outcomes were work-to-family and family-to-work conflict and depressive symptoms and anxiety symptoms during August 2020. Depressive symptoms between 2018 (before the COVID-19 pandemic) and 2020 (during the COVID-19 pandemic) were compared by gender. Among 215 physician parents who completed the August 2020 survey, 114 (53.0%) were female and the weighted mean (SD) age was 40.1 (3.57) years. Among physician parents, women were more likely to be responsible for childcare or schooling (24.6% [95% CI, 19.0%-30.2%] vs 0.8% [95% CI, 0.01%-2.1%]; P < .001) and household tasks (31.4% [95% CI, 25.4%-37.4%] vs 7.2% [95% CI, 3.5%-10.9%]; P < .001) during the pandemic compared with men. Women were also more likely than men to work primarily from home (40.9% [95% CI, 35.1%-46.8%] vs 22.0% [95% CI, 17.2%-26.8%]; P < .001) and reduce their work hours (19.4% [95% CI, 14.7%-24.1%] vs 9.4% [95% CI, 6.0%-12.8%]; P = .007). Women experienced greater work-to-family conflict (β = 2.79; 95% CI, 1.00 to 4.59; P = .03), family-to-work conflict (β = 3.09; 95% CI, 1.18-4.99; P = .02), and depressive (β = 1.76; 95% CI, 0.56-2.95; P = .046) and anxiety (β = 2.87; 95% CI, 1.49-4.26; P < .001) symptoms compared with men. We observed a difference between women and men in depressive symptoms during the COVID-19 pandemic (mean [SD] PHQ-9 score: 5.05 [6.64] vs 3.52 [5.75]; P = .009) that was not present before the pandemic (mean [SD] PHQ-9 score: 3.69 [5.26] vs 3.60 [6.30]; P = .86). This study found significant gender disparities in work and family experiences and mental health symptoms among physician parents during the COVID-19 pandemic, which may translate to increased risk for suicide, medical errors, and lower quality of patient care for physician mothers. Institutional and public policy solutions are needed to mitigate the potential adverse consequences for women's careers and well-being.