There are four distinct runs of Chinook Salmon (Oncorhynchus tshawytscha) in the Central Valley, named after their primary adult return times: fall, late-fall, winter, and spring run. Estimating the run-specific composition of juveniles entering and leaving the Sacramento–San Joaquin Delta is crucial for assessing population status and processes that affect juvenile survival through the Delta. Historically, the run of juvenile Chinook Salmon captured in the field has been determined using length-at-date criteria (LDC); however, LDC run assignments may be inaccurate if there is high overlap in the run-specific timing and size of juveniles entering and leaving the Delta. In this study, we use genetic run assignments to assess the accuracy of LDC at two trawl locations in the Sacramento River (Delta entry) and at Chipps Island (Delta exit). Fin tissues were collected from approximately 7,500 juvenile Chinook Salmon captured in trawl samples between 2007 and 2011. Tissues were analyzed using 21 microsatellites to determine genetic run assignments for individuals, which we compared with LDC run assignments. Across years, there was extensive overlap among the distributions of run-specific fork lengths of genetically identified juveniles, indicating that run compositions based on LDC assignments would tend to underestimate fall-run and especially late-fall-run compositions at both trawl locations, and greatly overestimate spring-run compositions (both locations) and winter-run compositions (Chipps Island). We therefore strongly support ongoing efforts to include tissue sampling and genetic run identification of juvenile Chinook Salmon at key monitoring locations in the Sacramento–San Joaquin River system.