© 2018 Taylor & Francis Group, LLC Background: Non-binary gender measurement has grown out of a need for accurate representation in scholarship and public health services available to a diverse gender population. Aims: The Genderqueer Identity Scale (GQI) was developed to allow for a multidimensional assessment of genderqueer identity, including non-binary identity, socially constructed versus essentialist gender, theoretical awareness of gender concepts, and gender fluidity. The GQI was designed to assess gender identity across a full spectrum of gender, at any age after mid-adolescence, and at various stages of gender identity development, including prior to, during, and after a gender transition, where applicable. Two of the GQI subscales focus on intrapersonal processes, while two focus on interpersonal processes. Methods: The measure was piloted and refined across four distinct samples: a U.S. university based LGBT sample, consecutive clinical referrals at the Center of Expertise on Gender Dysphoria in Amsterdam, the Netherlands, a Dutch LGB community sample, and an online survey forum (LGBTQ). Results: The first exploratory factor analysis identified minor potential adjustments, which were refined and retested. Researchers evaluated and cross-validated the hypothesized factor structure and determined that the three factor GQI subscales and the unidimensional Gender Fluidity measure yielded internally consistent and valid scores among transgender individuals seeking clinical treatment and LGB individuals within a community setting. The exploratory and confirmatory factor analyses provide evidence of good reliability, construct validity, and internal consistency of all four subscales. Discussion: The subscales were appropriate across a spectrum of gender identities and can be taken in the same form over time and across gender transition statuses, making them suitable for clinical evaluation and community based longitudinal research with trans-identified or gender nonconforming persons. The development of the GQI fills critical gaps in gender-related measurement including the ability to assess multiple dimensions of gender identity, and to assess gender identity across time.