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.