Background: Non-Alcoholic Fatty Liver Disease (NAFLD) is a common co-morbidity with obesity. Recent studies in adults with obesity have shown an association between subclinical hypothyroidism (SH) and NAFLD. Few studies of children from Europe have also assessed the association of SH with NAFLD diagnosed by ultrasound (1). Little is known about this association in children from underrepresented minorities in the US, especially in children of Latina ancestry and those with biopsy-proven NAFLD. Thyroid hormone impacts major metabolic pathways and overt hypothyroidism has been associated with insulin resistance and dyslipidemia. Aim: To assess the prevalence of SH in children of primarily Latina ancestry with biopsy-proven NAFLD as compared to children of the same age range visiting the clinic during the same years. Methods: We performed a retrospective study of 4363 children aged 5-19 years seen in an urban ambulatory care setting primarily serving children of Latina ancestry (> 80%). Sixty-three of these children had NAFLD proven by liver biopsy. Results of thyroid, liver, and lipid studies as well as demographic data were extracted from the electronic health record. A descriptive evaluation was performed. TSH values were divided into quartiles: Q1: TSH ≤1.07 mIU/L, Q2: TSH 1.07-1.59 mIU/L, Q3: TSH 1.59-2.36 mIU/L, Q4: TSH ≥ 2.36 mIU/L. The association of TSH quartiles with NAFLD was assessed by univariate analysis and multivariate logistic regression adjusting for age, gender, and severity of obesity. All analyses were performed in R statistical software. Results: There was a higher proportion of boys (74% vs. 40%, p < 0.001) in those with NAFLD, and they were younger (12.9±3.1 vs. 14.7±3.7 years, p < 0.001). Children with biopsy-proven NAFLD had significantly higher BMI, TSH, and ALT when compared to those without NAFLD (TSH, cases: 2.9 ± 1.2 mIU/L, controls: 1.9 ± 1.2 mIU/L, p<0.0001). The proportion of children with abnormal ALT, AST, and GGT was significantly higher in those with NAFLD compared to the controls (p < 0.001). They also had a higher proportion of abnormal total, HDL, and non-HDL cholesterol (p = 0.01). The odds of having NAFLD was statistically higher for children in Q3 (OR 5.07, p=0.003) and Q4 (OR 8.48, p<0.0001) of TSH, compared to those in Q1. After adjusting for age, gender, and severity of obesity, biopsy-proven NAFLD approached significantly higher odds (2.7, p = 0.07) in Q3 and was statistically significant in Q4 (OR= 4.0, p=0.0113). Conclusion: Children of Latina ancestry with NAFLD diagnosed by histology are at higher odds of having SH. Based on the results of this study, we hypothesize that thyroid dysfunction plays a mediator role in the development of NAFLD and should be investigated in all children with obesity.