Transition from elementary to middle school is commonly seen as a period of stress, impacting students’ school adjustment. The present longitudinal study aimed to analyze the difference in stress levels between the end of 4th grade and 5th grade, while also analyzing gender differences and 5th grade retention. Two hundred fifty-eight 4th grade students (Mage = 9.55; SD = 0.77) from six Portuguese public schools, from the municipality of Torres Vedras, participated in this study. Self-report questionnaires were administered at the end of the 4th and 5th grades, and 5th grade school records were also collected. Results showed that 5th graders present higher levels of Academic Stress (d = .29) and Teacher/Rules Stress (d = .28). Girls had a greater increase of Peer-related Stress with the transition (p < .01). Students who were retained at the end of 5th grade showed higher increases of Teacher/Rules Stress (p < .05). Intervention programs that aim to support the transition from elementary to middle school are needed, in order to reduce the increase of stress levels at 5th grade and to promote a better school adjustment in the first year of middle school.