This article analyzes the effect of classroom separation of twins on their cognitive abilities, measured at different ages in Dutch primary education. We use a large longitudinal school-based sample of twins and their classmates. The analysis tries to reduce the bias by unobserved factors due to the nonrandom assignment of twins by taking into account differences in school environment, previous test scores and variation in class assignment between years. We find that classroom separation matters for language in Grade 2. Nonseparated twins score higher on language, and the difference is larger for same-sex pairs. This finding is robust for various methods that take unobserved effects into account. In addition, there is some evidence for higher scores in arithmetic in Grade 2. For the higher grades we find no effect of classroom separation on cognitive ability. In the analysis of the effect of a separation of at least 3 years we find that separation increases language performance between Grade 6 and 8 for opposite-sex pairs.