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Purpose Misgendering of transgender and non-binary (TGNB) individuals in healthcare settings can lead to worsened mental and physical health outcomes and decreased utilisation of care. Few studies have investigated the factors that contribute to this phenomenon. The purpose of this study was to apply qualitative methods to explore sources of misgendering, its perceived impact, prevention strategies and clinician responses to accidentally misgendering a patient, as identified by TGNB patients and gender-affirming care clinicians.Methods Between April and June 2022, 20 semi-structured interviews were performed at an academic medical centre in Southern California. Participants were recruited via purposive sampling and included: (1) TGNB patients (n=8) recruited from an interdisciplinary gender-affirming urological practice and (2) gender-affirming care clinicians (n=12) recruited from a regional interdisciplinary Gender Health conference, three of whom identified as TGNB. Interviews were conducted in person or virtually using an open-ended topic guide, audio recorded and transcribed verbatim. Inductive thematic analysis was performed by two independent study personnel who hand-coded the transcripts.Results Four overarching themes were identified: (1) misgendering originates from multiple sources, (2) misgendering discourages individual access to healthcare, creates community hesitation and its perceived impact is modified by setting and intentionality, (3) building a gender-affirming healthcare system requires integration of behaviour, policy and technology and (4) clinicians respond to accidental misgendering by acknowledging, apologising, advancing and acting.Conclusion Our data suggest that misgendering arises from both interpersonal communication and structural factors within healthcare systems, leading to perceived harm and diminished TGNB access to health services. Any potential solution to reduce this phenomenon will require a multifaceted approach integrating behavioural, technological and institutional policy strategies with system-level implementation efforts.