OBJECTIVE: To evaluate the performance of a cell-free DNA (cfDNA) assay that uses next-generation sequencing with quantitative counting templates for the clinical detection of the fetal RHD genotype in a diverse RhD-negative pregnant population in the United States. METHODS: This retrospective cohort study was conducted in four U.S. health care centers. The same next-generation sequencing quantitative counting template cfDNA fetal RhD assay was offered to nonalloimmunized RhD-negative pregnant individuals as part of clinical care. Rh immune globulin (RhIG) was administered at the discretion of the clinician. The sensitivity, specificity, and accuracy of the assay were calculated considering the neonatal RhD serology results. RESULTS: A total of 401 nonalloimmunized RhD-negative pregnant individuals who received clinical care in the period from August 2020 to November 2023 were included in the analysis. The D antigen cfDNA result was 100% concordant with the neonatal serology, resulting in 100% sensitivity, 100% positive predictive value (95% CI, 98.6-100% for both), 100% specificity, and 100% negative predictive value (95% CI, 97.4-100% for both). There were 10 pregnant individuals in whom the cfDNA analysis identified a non- RHD gene deletion, including RhDΨ (n=5) and RHD-CE-D hybrid variants (n=5). Rh immune globulin was administered antenatally to 93.1% of pregnant individuals, with cfDNA results indicating an RhD-positive fetus compared with 75.0% of pregnant individuals with cfDNA results indicating an RhD-negative fetus, signifying that clinicians were using the cfDNA results to guide pregnancy management. CONCLUSION: This next-generation sequencing with quantitative counting templates cfDNA analysis for detecting fetal RhD status is highly accurate with no false-positive or false-negative results in 401 racially and ethnically diverse pregnant individuals with 100% follow-up of all live births. This study and prior studies of this assay support a recommendation to offer cfDNA screening for fetal Rh status as an alternative option to prophylactic RhIG for all nonalloimmunized RhD-negative individuals, which will result in more efficient and targeted prenatal care with administration of RhIG only when medically indicated.