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Background: The Undetectable=Untransmittable (U=U) message has been promoted since it was demonstrated that viral suppression through HIV treatment prevents sexual transmission between serodiscordant partners (HIV treatment as prevention). Our study assessed familiarity with, perceived accuracy of, and willingness to rely on U=U in a national sample of gay and bisexual men in Australia. Methods: We conducted a national, online cross-sectional survey in April-June 2021. Eligible participants were gay, bisexual and queer men and non-binary people who lived in Australia. Logistic regression was used to identify factors associated with familiarity, perceived accuracy and willingness to rely on U=U (by having condomless sex with a partner with HIV who has an undetectable viral load). Results: Of 1280 participants, most were familiar with U=U (1006/1280 78.6%), the majority of whom believed U=U was accurate (677/1006 67.3%). Both familiarity and perceived accuracy were higher among participants living with HIV, followed by pre-exposure prophylaxis (PrEP) users, HIV-negative participants not taking PrEP, and untested/unknown status participants. Knowing at least one person living with HIV, among other factors, was associated with familiarity and perceived accuracy of U=U and familiarity was associated with perceived accuracy. Among participants familiar with U=U, less than half were willing to rely on U=U (473/1006 47.0%). Familiarity with U=U and knowing at least one person living with HIV were associated with willingness to rely on U=U, among other factors. Conclusions: We found familiarity with U=U was associated with perceived accuracy and willingness to rely upon it. There is an ongoing need to educate gay and bisexual men (particularly HIV-negative men) about U=U and its benefits.