This study conducted among transgender women in São Paulo, Brazil assessed the acceptability and suitability of screening sexually transmitted infections (STIs), such as Chlamydia trachomatis and Neisseria gonorrhoeae, by sampling multiple anatomical sites (i.e. urethral, anorectal, oropharyngeal, and neovaginal), and utilizing self- or provider-collection methods. First, a convenience sample of 23 cohort participants were recruited during a scheduled study visit between October and November 2018. Data collection was through a short investigator-led quantitative survey in Portuguese, and included presentation of investigator-designed, gender-neutral instructional diagrams to guide self-sampling. Three supplemental focus group discussions (FGDs) with a total of 30 participants guided by semi-structured script were conducted in Portuguese between September and October 2019. All participants reported being assigned male sex at birth and self-identified with a feminine gender identity at time of study. All survey respondents (100%; n = 23) indicated willingness to provide samples for STI screening during a future study visit. Preference was for self-collection of urine samples (83%; n = 19), urethral swabs (82%; n = 18), and anorectal swabs (77%; n = 17). A lower preference for self-collection of oropharyngeal swabs (48%; n = 11) was observed. Most respondents (78%; n = 18) indicated that they would not prefer specimens to be collected by a health professional, mainly due to 'more privacy' (72%; n = 13). All respondents indicated that they would feel comfortable to provide a self-collected sample based on instructional diagrams shown. In FGDs, although the collection by a health professional was described as a technically safer option for some participants, there was a preference for self-collection to avoid discomfort and embarrassment in exposing the body. Overall, this sub-study suggested acceptability among transgender women of introducing self-sampling for etiological diagnosis of STIs from potential infection sites. Uptake and usability will be explored further in a cross-sectional STI prevalence study of transgender women in Brazil.