Although female representation has increased in clinical trials, little is known about how clinical trial representation compares with burden of disease or is associated with clinical trial features, including disease category. To describe the rate of sex reporting (ie, the presence of clinical trial data according to sex), compare the female burden of disease with the female proportion of clinical trial enrollees, and investigate the associations of disease category and clinical trial features with the female proportion of clinical trial enrollees. This cross-sectional study included descriptive analyses and logistic and generalized linear regression analyses with a logit link. Data were downloaded from the Aggregate Analysis of ClinicalTrials.gov database for all studies registered between March 1, 2000, and March 9, 2020. Enrollment was compared with data from the 2016 Global Burden of Disease database. Of 328 452 clinical trials, 70 095 were excluded because they had noninterventional designs, 167 936 because they had recruitment sites outside the US, 69 084 because they had no reported results, 1003 because they received primary funding from the US military, and 314 because they had unclear sex categories. A total of 20 020 interventional studies enrolling approximately 5.11 million participants met inclusion criteria and were divided into those with and without data on participant sex. The primary exposure variable was clinical trial disease category. Secondary exposure variables included funding, study design, and study phase. Sex reporting and female proportion of participants in clinical trials. Among 20 020 clinical trials from 2000 to 2020, 19 866 studies (99.2%) reported sex, and 154 studies (0.8%) did not. Clinical trials in the fields of oncology (46% of disability-adjusted life-years [DALYs]; 43% of participants), neurology (56% of DALYs; 53% of participants), immunology (49% of DALYs; 46% of participants), and nephrology (45% of DALYs; 42% of participants) had the lowest female representation relative to corresponding DALYs. Male participants were underrepresented in 8 disease categories, with the greatest disparity in clinical trials of musculoskeletal disease and trauma (11.3% difference between representation and proportion of DALYs). Clinical trials of preventive interventions were associated with greater female enrollment (adjusted relative difference, 8.48%; 95% CI, 3.77%-13.00%). Clinical trials in cardiology (adjusted relative difference, -18.68%; 95% CI, -22.87% to -14.47%) and pediatrics (adjusted relative difference, -20.47%; 95% CI, -25.77% to -15.16%) had the greatest negative association with female enrollment. In this study, sex differences in clinical trials varied by clinical trial disease category, with male and female participants underrepresented in different medical fields. Although sex equity has progressed, these findings suggest that sex bias in clinical trials persists within medical fields, with negative consequences for the health of all individuals.