Background Diagnostic errors cause substantial preventable harm, but national estimates vary widely from 40,000 to 4 million annually. This cross-sectional analysis of a large medical malpractice claims database was the first phase of a three-phase project to estimate the US burden of serious misdiagnosis-related harms. Methods We sought to identify diseases accounting for the majority of serious misdiagnosis-related harms (morbidity/mortality). Diagnostic error cases were identified from Controlled Risk Insurance Company (CRICO)’s Comparative Benchmarking System (CBS) database (2006–2015), representing 28.7% of all US malpractice claims. Diseases were grouped according to the Agency for Healthcare Research and Quality (AHRQ) Clinical Classifications Software (CCS) that aggregates the International Classification of Diseases diagnostic codes into clinically sensible groupings. We analyzed vascular events, infections, and cancers (the “Big Three”), including frequency, severity, and settings. High-severity (serious) harms were defined by scores of 6–9 (serious, permanent disability, or death) on the National Association of Insurance Commissioners (NAIC) Severity of Injury Scale. Results From 55,377 closed claims, we analyzed 11,592 diagnostic error cases [median age 49, interquartile range (IQR) 36–60; 51.7% female]. These included 7379 with high-severity harms (53.0% death). The Big Three diseases accounted for 74.1% of high-severity cases (vascular events 22.8%, infections 13.5%, and cancers 37.8%). In aggregate, the top five from each category (n = 15 diseases) accounted for 47.1% of high-severity cases. The most frequent disease in each category, respectively, was stroke, sepsis, and lung cancer. Causes were disproportionately clinical judgment factors (85.7%) across categories (range 82.0–88.8%). Conclusions The Big Three diseases account for about three-fourths of serious misdiagnosis-related harms. Initial efforts to improve diagnosis should focus on vascular events, infections, and cancers.