Background Depression, anxiety, and stress symptoms are common among adolescents; however, studies exploring their relationship with chronotype in European youth populations is scarce. This study aimed to evaluate the association between chronotype status and depression, anxiety, and stress symptoms in adolescents. Methods A secondary analysis of the Eating Healthy and Daily Life Activities (EHDLA) cross-sectional study was performed in 703 adolescents (56.3% girls) between 12 and 17 years from the Valle de Ricote, Spain. Chronotype preference was assessed using the Morningness/Eveningness Scale in Children (MESC), while symptoms were evaluated using the Depression, Anxiety, and Stress Scale (DASS-21). A robust generalized linear regression model was used to evaluate the associations between chronotype prefernces and symptoms of depression, anxiety and stress in adolescents. Results After adjusting for potential covariates (sex, age, socioeconomic status, body mass index, sleep duration, physical activity, sedentary behavior, and energy intake), the highest probability of having depression, anxiety, and stress was identified in those with an eveningness chronotype preference (depression: 27.4%, 95% confidence interval [CI] 17.5-40.1%; anxiety: 28.5%, 95% CI 18.6-41.0%; stress: 47.6%, 95% CI 34.1-61.5%). Conversely, the lowest probability was observed in adolescents with a morningness chronotype preference (depression: 11.9%, 95% CI 8.3-16.8%; anxiety: 15.4%, 95% CI 11.2-28.9%; stress: 19.5%, 95% CI 14.7-25.5%). Significant differences were found when comparing participants with the eveningness chronotype to those with a morningness or intermediate chronotype preference (p < 0.05 for all comparisons). Conclusions Depression, anxiety, and stress symptoms were more likely in adolescents with an eveningness chronotype preference than in those with morningness or intermediate chronotypes. Chronotype preferences should be taken into account for developing interventions that promote better mental health and healthy sleep habits in adolescents.