Prejudice is a structural determinant of transgender health, yet the field still lacks measurement instruments that distinguish attitudes toward transgender women, transgender men, and nonbinary people, that have been developed with input from transgender people themselves, and that are available in the working languages of the Global South. We address these gaps by developing and validating the Prejudice against Transgender Scales (PTS), a family of three parallel 10-item measures, in Brazil, Mexico, and South Africa, and in Portuguese, Spanish, and English. Eight studies (N = 1,618) provide evidence of content validity through expert evaluation, transgender community consultation, and respondent comprehensibility testing (Studies 1a–1c); a unifactorial structure with strong item-response-theory discrimination parameters (Study 2); confirmatory factor structure and measurement invariance across respondent gender and age (Study 3); convergent and discriminant validity (Study 4); and configural and metric invariance across the three national and linguistic contexts, alongside excellent internal consistency in every country and language (Study 5). South Africa exhibited higher mean prejudice than Brazil and Mexico across all three targets, and in each country the PTS predicted opposition to gender-affirming healthcare policies. Finally, a random-effects meta-analytic synthesis of the gender effects across Studies 2 to 5 (Study 6) shows that cisgender heterosexual men (vs. women) consistently expressed more prejudice on all three target groups, with pooled ds of 0.53 (PTS-W), 0.51 (PTS-M), and 0.57 (PTS-NB). The PTS connects the social-psychological measurement of prejudice to a salient public-health outcome and gives researchers, clinicians, and advocates a culturally informed and linguistically validated tool for the study and reduction of transgender health inequity.