Abstract Background: Metabolic-associated steatotic liver disease (MASLD) is a prevalent chronic liver condition with an increased risk of cancer-related mortality. In Latin America, MASLD prevalence is estimated at 40%, with Chile reporting rates of 47.5% in 2019. Early detection is crucial for MASLD control and management. Liver biopsy, the gold standard for diagnosis, is invasive and costly. Ultrasound (US) is a preferred alternative, yet its precision varies depending on the observer interpretation. Methods: We conducted a cross-sectional analysis of a subset of baseline US images from the Chile Biliary Longitudinal Study (Chile BiLS), a cohort of women aged 50-74 with gallstones in Chile. Of the 4,032 participants recruited from Cautín Province in the Araucanía region, a random sample of 10% of the images were selected for review by a radiologist from Baylor College of Medicine (Observer 1) and compared to the original readings generated at the baseline exam by a team of Chile BiLS ultrasound technicians. To assess reproducibility, 33 blinded duplicate images from the 10% sample were reanalyzed by Observer 1 and an additional Chile BiLS technician who was not part of the original baseline team (Observer 2). Inter- and intra-observer agreement was assessed using weighted kappa values. Images were categorized as none, mild, moderate/severe. Results: Of the 409 images reviewed, agreement between Observer 1 and original readings across all categories of steatosis was poor (kappa: 0.09; CI 95% 0.06;0.11, p <0.001). The 33 duplicates compared to the original readings showed slight agreement for Observer 1 (kappa: 0.11 CI 95% 0.02;0.20, p <0.05) and fair for Observer 2 (kappa: 0.28 (CI 95% 0.07 - 0.49, p <0.001). The intra-observer agreement for Observer 1 was moderate, with a kappa of 0.41 (CI 95% 0.09 - 0.74, p <0.05), and for Observer 2, it was substantial with a weighted kappa of 0.69 (CI 95% 0.5 - 0.9, p <0.001). Conclusion: The study highlights the variability in US interpretation of hepatic steatosis, emphasizing the importance of standardized training and methodology. US's sensitivity and specificity is operator dependence, which impacts the diagnostic interpretation. Our findings underscore the significance of inter and intra-observer comparisons for optimal diagnosis, especially when US is the primary diagnostic tool in MASLD assessment. Finally, our results emphasize the importance of group training. Observer 2, who underwent the same training as the original Chile BiLS ultrasound technicians and was consistently exposed to this technique as a diagnostic method, exhibited more consistent results. This reinforces the importance of collective training initiatives to enhance the reliability and accuracy of US in MASLD diagnosis. Citation Format: Maria Spencer Sandino, Vanessa Van De Wyngard, David Wynne, Maya Balakrishnan, Noldy Mardones, Ruth Pfeiffer, Allan Hildesheim, Catterina Ferreccio, Jill Koshiol. Inter-observer agreement in ultrasound-detected steatotic liver in the Chile Biliary Longitudinal Study (BiLS) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6083.