Abstract Body: Childhood-onset essential hypertension (COEH) is characterized by elevated blood pressure of unknown etiology diagnosed before age 18. COEH affects ~2.1% of children in the United States (U.S.), with a higher prevalence among those of self-reported African ancestries. The genetic underpinnings of COEH are unknown, but its early onset, high heritability, low correlation with environmental factors, and low prevalence suggest a role for monogenic variation. To investigate evidence of Mendelian inheritance in COEH, we performed genome sequencing in 207 individuals from the U.S., including 73 probands and their relatives. We identified six candidate genes with strong evidence of involvement in hypertension in ten families, including SYNE1 (three families), HMCN1 (two), TNS1 (two), FBN2 (one), THBS2 (one), and LRRFIP1 (one). Additionally, six genes with moderate evidence of involvement in hypertension were identified in eight families. Implicated variants in all candidate genes showed evidence of autosomal recessive inheritance, with the resulting candidate genes predominantly expressed in vessels and enriched in individuals of African ancestry. All identified variants were predicted to be damaging to the resulting protein by multiple in silico tools and were either novel or exceedingly rare in population databases. By sequencing an additional 88 probands from South Africa with COEH, we found that 25% (22/88) and 9% (8/88) of them carried at least two putatively damaging variants in SYNE1 and HMCN1, respectively. Leveraging data from the All of Us initiative, we found that individuals with a history of pediatric essential hypertension are more likely to have damaging variants in our strong candidates as compared to individuals with no history of hypertension in childhood. Notably, SYNE1, HMCN1, and TNS1 are presumed to play a role in activating Rho-kinase (ROCK), a protein that regulates contractility in vascular smooth muscle cells (VSMCs). Reduced expression of strong candidates thus far assessed (i.e., SYNE1, HMCN1) leads to an abnormal VSMCs phenotype that can be rescued by a downstream ROCK pathway inhibitor. Our findings suggest that genes associated with COEH primarily regulate vascular tone, offering insights that could inform the development of targeted therapeutics and promote precision medicine in the future.