Zygosity determination using similarity ratings is frequently applied in twin studies. A correct determination of zygosity is essential for the estimation of heritability and environmental influences on phenotypes. Therefore, the present study examined the validity of two similarity questionnaires used in the German TwinLife study, in which data from 4,097 twin pairs and their families were assessed: twin children’s zygosities were determined with the Zygosity Questionnaire for Young Twins, which was administered in parent-report form. For adolescent twins, the Self Report Zygosity Questionnaire was used. For the present validation analyses, DNA samples of N = 328 twin pairs were collected via buccal swabs. In this DNA subsample, questionnaires were filled out by parents for n = 212 (aged 4 to 12 years) twin pairs while self-reports were collected from n = 116 adolescent twins (16 to 23 years of age). Using DNA-based zygosity as criteria, correct classification rates of 97% for parent- and 92% for self-reports were established and cross-validated. Additionally, classification rates based on a single item and variants of questionnaire based zygosity determination used in other twin studies were calculated and compared. Implications of incorrectly classified zygosity on genetic and environmental estimates in twin studies are discussed.