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Background: Small intestinal bacterial overgrowth (SIBO) has been associated with small intestinal and colonic surgeries. However, with few large clinical trials and potential confounding by factors such as PPI use, the role of upper gastrointestinal surgeries in clinically significant SIBO remains unclear. Patients with prior Roux-en-Y gastric bypass (RYGB) frequently present with symptoms similar to SIBO, such as nausea, vomiting, bloating, gas, diarrhea, and abdominal pain that may lead to reconstructive surgery. Aim: To identify the risk of SIBO among RYGB patients using lactulose breath test (LBT). Methods: This was a retrospective cohort study of 313 symptomatic patients referred to a tertiary care center for LBT. Conditions and use of medications thought to be associated with SIBO (bowel surgery, diabetes, irritable bowel syndrome, pancreatic insufficiency, thyroid disorder, proton pump inhibitor [PPI] use) and presenting symptoms were reviewed. Patients with prior RYGB were identified. Positive LBT required a rise >20ppm in breath hydrogen or methane concentration within 60 minutes. Univariate and multivariate analyses were performed using Chi-square or Fisher-exact test and logistic regression, respectively. Statistical significance was established using p<0.05. Results: 105 (35.5%) patients had positive LBT. Overall, 25 patients had prior RYGB. Univariate analysis revealed an increased rate of positive LBT among RYGB patients compared to controls (70.0% vs 34.0%, OR=4.54, p=0.02). On multivariate analysis, RYGB remained independently associated with positive LBT when other risk factors for SIBO were controlled (OR=4.35, p=0.04). Secondary analysis by adding an interaction term for PPI use and RYGB to the multivariate analysis demonstrated no effect modification by PPI on RYGB’s risk for SIBO. A separate multivariate analysis was performed using RYGB and presenting symptoms as predictors. RYGB remained an independent risk factor for positive LBT when presenting symptoms were controlled. Conclusions: RYGB increased the risk for symptomatic SIBO. When other factors associated with SIBO were controlled, RYGB remained an independent risk factor for SIBO. In particular, PPI use, which was previously identified as a strong SIBO risk factor and is widely used in RYGB patients, did not significantly confound or modify the effect of RYGB on SIBO. When evaluating RYGB patients with post-operative abdominal symptoms, SIBO should be considered prior to pursuing more invasive diagnostic and therapeutic procedures. Further prospective studies are needed to better identify the RYGB patients most at risk for SIBO and formulate the optimal treatment strategy in this patient group.