Introduction: Transfeminine women in South Africa have a high HIV risk due to structural, behavioural, and psychosocial factors. Transfeminine women and feminine identifying men who have sex with men (MSM) are often conflated or grouped with transgender or MSM categories in HIV service programming, although they don't necessarily identify as either. We aimed to investigate gender expression among feminine identifying people who were assigned male at birth. We examined how local conceptualizations of sexuality and gender intersect with the key population label of 'transgender' imported into local HIV programming. Methods: A qualitative cohort nested within the HPTN 071 (PopART) trial included longitudinal, in-depth interviews with eight transfeminine women (four who disclosed as living with HIV). Data were collected approximately every six weeks between January 2016 and October 2017. We discuss gender identification presented in participants' daily lives and in relation to HIV service access. Results: Of the eight participants, only one accepted 'transgender' as a label, and even she used varying terms at different times to describe her identity. For participants, a feminine identity included dressing in normatively feminine clothes; using feminine terms, pronouns and names; and adopting stereotypically feminine mannerisms. Participants would switch between typically feminine and masculine norms in response to contextual cues and audience. For example, some participants accepted identification as masculine gay men amongst their family members, but amongst peers, they expressed a more effeminate identity and with partners they took on a feminine identity. Conclusions: Our findings are amongst the first exploratory and descriptive data of transfeminine women in South Africa. We show how transfeminine women navigate fluid gender identities that could pose a challenge for accessing and utilizing HIV services that are currently set up for transgender individuals or MSM. More work needs to be done to understand and respond to the diverse and shifting ways people experience their gender identities in this high HIV burden context.