This study investigated the development of family relationships in lesbian families who conceived after donor insemination (DI). The main characteristics of this unknown family structure were analysed. An anonymous donor was used to conceive, the family unit consisted of two mothers and a father was non-existent, and both women had a homosexual orientation. A total of 50 lesbian couples who conceived after DI with children aged between 1 and 2 years participated in this study, using a standardized interview created for this study. After the birth of their child, 56% of the lesbian mothers (n = 100) would have wanted the identity of the donor to be registered, while 10% would have done so at the time of the insemination. Opinions differed in 12 of the 50 couples: the biological mother was in favour of identity registration while the social mother was not. Both women considered themselves a parent of the child with equally shared responsibilities. Mothers were open about the special features of their family structure with children and within their immediate social network. Only 30% disclosed their lesbian identity in a broader social environment. The features of these newly created families may influence the psychological development of the children and therefore make long-term follow-up studies indispensable.