Trans masculine, two-spirit, and non-binary people who are gay, bisexual or otherwise have sex with men (TGBM) are under-tested for sexually transmitted infections (STI) and may face complex, intersectional barriers that prevent them from accessing STI testing. As part of a study on gay, bisexual and other men who have sex with men’s (GBM) experiences of current STI testing systems in Ontario, Canada, this paper reports on the findings from TGBM participants’ experiences with in-person STI testing in a range of venues (i.e. Family doctors, walk-in clinics, and community-based organizations) to explore testing barriers specific to TGBM. Using a community-based research approach, between June 2020 and December 2021 peer researchers who identified as GBM conducted focus groups and interviews with 38 cis and trans GBM, 13 of whom identified as TGBM. Data were analyzed following grounded theory. When questioned about past experiences with testing, TGBM participants reported several barriers to STI testing within current testing models in Ontario due to cisnormativity and heteronormativity. Cisnormativity is the assumption that everyone identifies as the gender they were assigned at birth, and those who do not are considered “abnormal”, while heteronormativity is when it is assumed that everyone is heterosexual. From our research we identified three overarching themes concerning testing barriers among TGBM participants: (1) non-inclusive clinic environments, (2) lack of provider knowledge and competency, and (3) legal documentation. Inherent cis and heteronormativity in healthcare institutions appear to be factors shaping the historical under-testing for STI in the TGBM population. These findings suggest the relevance of implementing trans-specific clinical practices that reduce the stigma and barriers faced by TGBM in STI testing contexts, including: hosting all-gender testing hours, opening more LGBTQ+ clinics, offering training in transgender health to testing providers, and conducting a review of how gender markers on health documents can be more inclusive of trans, two-spirit, and non-binary communities.