Introduction: Cumulative evidence demonstrates that bisexual individuals face significant health disparities relative to heterosexual and gay/lesbian individuals. Researchers have hypothesized that these differences may be fueled by negative attitudes, prejudice, stigma, and discrimination toward bisexual individuals from heterosexual gay/lesbian individuals. Previous studies of attitudes toward bisexual individuals in the United States have been based almost exclusively on convenience samples, with limited generalizability. Methods: Data were collected from the 2015 National Survey of Sexual Health and Behavior (NSSHB), via an online questionnaire to a nationally-representative sample of self-identified heterosexual, gay/lesbian, asexual, and "other" adults (18 and over) in the U.S. We included two modified 5-item versions of the BIAS, validated scales that measure attitudes toward bisexual men and women. Data were analyzed using descriptives, gamma regression, and paired t-tests. Results: Age, sexual identity, gender, race/ethnicity, income, and educational attainment were all significantly associated with participants' attitudes toward bisexual individuals. Across sexual identities, self-identified "other" participants reported the most positive attitudes and heterosexual men reported the least positive attitudes. Overall, attitudes toward bisexual men were less positive than toward bisexual women across identities. Conclusions: As in research on convenience samples, we found a wide range of demographic characteristics were related with attitudes toward bisexual individuals in a nationally-representative sample of heterosexual, gay/lesbian, asexual, and "other"-identified adults in the U.S. Findings document the persistence of negative attitudes toward bisexual individuals and the need for developing intervention approaches to promote positive attitudes and tolerance toward bisexual individuals among heterosexual and gay/lesbian individuals and communities.