Nonalcoholic fatty liver disease (NAFLD) is the most common cause of liver disease, affecting approximately 3 in 10 obese children worldwide.We aimed to investigate the potential relationship between gut microbiota and NAFLD in obese youth, while considering the role of PNPLA3 rs738409, a strong genetic contributor to NAFLD.In this cross-sectional study, participants completed abdominal MRI to measure hepatic fat fraction (HFF), oral glucose tolerance test, and PNPLA3 rs738409 genotyping. Fecal samples were collected to analyze the V4 region of the 16S rRNA gene for intestinal bacteria characterization.Yale Pediatric Obesity Clinic.Obese youth (BMI > 95th percentile) with NAFLD (HFF ≥ 5.5%; n=44) and without NAFLD (HFF < 5.5%; n=29).Shannon-Wiener diversity index values and proportional bacterial abundance by NAFLD status and PNPLA3 genotype.Subjects with NAFLD had decreased bacterial alpha-diversity compared to those without NAFLD (p=0.013). Subjects with NAFLD showed a higher Firmicutes to Bacteroidetes (F/B) ratio (p=0.019) and lower abundance of Bacteroidetes (p=0.010), Prevotella (p=0.019), Gemmiger (p=0.003), and Oscillospira (p=0.036). F/B ratio, Bacteroidetes, Gemmiger, and Oscillospira were associated with HFF when controlling for group variations. We also observed an additive effect on HFF by PNPLA3 rs738409 and Gemmiger, and PNPLA3 rs738409 and Oscillospira.Obese youth with NAFLD have a different gut microbiota composition than those without NAFLD. These differences were still statistically significant when controlling for factors associated with NAFLD, including PNPLA3 rs738409.