Purpose:We report the diagnostic yield of whole-exome sequencing (WES) in 3,040 consecutive cases at a single clinical laboratory.Methods:WES was performed for many different clinical indications and included the proband plus two or more family members in 76% of cases.Results:The overall diagnostic yield of WES was 28.8%. The diagnostic yield was 23.6% in proband-only cases and 31.0% when three family members were analyzed. The highest yield was for patients who had disorders involving hearing (55%, N = 11), vision (47%, N = 60), the skeletal muscle system (40%, N = 43), the skeletal system (39%, N = 54), multiple congenital anomalies (36%, N = 729), skin (32%, N = 31), the central nervous system (31%, N = 1,082), and the cardiovascular system (28%, N = 54). Of 2,091 cases in which secondary findings were analyzed for 56 American College of Medical Genetics and Genomics-recommended genes, 6.2% (N = 129) had reportable pathogenic variants. In addition to cases with a definitive diagnosis, in 24.2% of cases a candidate gene was reported that may later be reclassified as being associated with a definitive diagnosis.Conclusion:Our experience with our first 3,040 WES cases suggests that analysis of trios significantly improves the diagnostic yield compared with proband-only testing for genetically heterogeneous disorders and facilitates identification of novel candidate genes.