We examined the item properties of the Two Peas Questionnaire (TPQ) among a sample of same-sex twin pairs from the Washington State Twin Registry. With the exception of the ‘two peas’ item, three of the mistakenness items showed differential item functioning. Results showed that the monozygotic (MZ) and dizygotic (DZ) pairs may differ in their responses on these items, even among those with similar latent traits of similarity and confusability. Upon comparing three classification methods to determine the zygosity of same-sex twins, the overall classification accuracy rate was over 90% using the unit-weighted pair zygosity sum score, providing an efficient and sufficiently accurate zygosity classification. Given the inherent nature of twin-pair similarity, the TPQ is more accurate in the identification of MZ than DZ pairs. We conclude that the TPQ is a generally accurate, but by no means infallible, method of determining zygosity in twins who have not been genotyped.