In recent years, epigenetic markers emerged as a new tool to understand the influence of lifestyle factors on obesity phenotypes. Adolescence is considered an important epigenetic window over a human's lifetime. The objective of this work was to explore baseline changes in DNA methylation that could be associated with a better weight loss response after a multidisciplinary intervention program in Spanish obese or overweight adolescents. Overweight or obese adolescents (n=107) undergoing 10 wk of a multidisciplinary intervention for weight loss were assigned as high or low responders to the treatment. A methylation microarray was performed to search for baseline epigenetic differences between the 2 groups (12 subjects/group), and MALDI‐TOF mass spectrometry was used to validate (n=107) relevant CpG sites and surrounding regions. After validation, 5 regions located in or near AQP9, DUSP22, HIPK3, TNNT1, and TNNI3 genes showed differential methylation levels between high and low responders to the multidisciplinary weight loss intervention. Moreover, a calculated methylation score was significantly associated with changes in weight, BMI‐SDS, and body fat mass loss after the treatment. In summary, we have identified 5 DNA regions that are differentially methylated depending on weight loss response. These methylation changes may help to better understand the weight loss response in obese adolescents.—Moleres, A., Campión, J., Milagro, F. I., Marcos, A., Campoy, C., Garagorri, J. M., Gómez‐Martínez, S., Martínez, J. A., Azcona‐Sanjulián, M. C., Martí, A. Differential DNA methylation patterns between high and low responders to a weight loss intervention in overweight or obese adolescents: the EVASYON study. FASEB J. 27, 2504–2512 (2013). http://www.fasebj.org