There is a need for ongoing behavioral surveillance of human immunodeficiency virus (HIV)/sexually transmitted infection (STI) risk among transgender women, using assessments adapted to this population. We therefore developed and piloted the Transgender Women's Internet Survey and Testing (TWIST) study, a cross-sectional behavioral survey of transgender women in the United States coupled with remote biospecimen collection and testing. Methods: Participants age 15 + were recruited by using social media advertisements. Participants were eligible to take the survey if they reported male sex at birth, identified as female or as a transgender woman, resided in the United States, and reported ever having oral, vaginal, or anal sex. We examined a number of behavioral indicators by age, county population density, and medical gender affirmation treatment, using multivariable regression modeling. A sample of respondents was invited to receive a home biospecimen collection kit for HIV/ STI testing. Results: The 401 participants were mainly non-Hispanic white and younger than 25 years. Self-reported HIV prevalence was 1.3% (5/401), and almost half (47.1%, 189/401) did not know their HIV status. Receiving medical gender affirmation was strongly associated with past-year HIV and STI testing, independent of general health care engagement. Of the 155 participants invited to receive home biospecimen collection kits, 48 (31.0%) consented and of those, 21 (43.8%) returned specimens for testing. Conclusion: This pilot study successfully reached its recruitment target and generated useful behavioral measures from an online sample of transgender women. We anticipate that online recruitment combined with selfcollection of biospecimens will serve as an innovative and scalable strategy for ongoing monitoring of HIV/STI behavioral trends among U.S. transgender women.