<p>The purpose of the present study was to examine and compare the attitudes of residents and seniors in family medicine from southern Israel toward transgender patients. It was a cross-sectional study based on a self-administered questionnaire. Attitudes toward transgender patients were assessed by the Transgender Attitudes and Beliefs Scale composed of “Interpersonal comfort,” “Sex/gender beliefs”, and “Human value” domains. The “Sex/gender beliefs” domain measures a person's belief about whether gender is a fixed, binary characteristic or a more fluid continuum. Fifty-five specialists (26; 47.3% males) and 56 residents (31; 56.4% males) took part in the study. Overall, family medicine physicians showed a favorable attitude toward transgender individuals on the “Human value” subscale with 82 (74%) giving favorable scores, but only 38 (34.2%) of the physicians demonstrated a favorable attitude on the “Sex/gender beliefs” scale. In the multivariable regression analysis, where age, sex, ethnicity, seniority, and level of religiosity were included as predictor variables, non-Jewish ethnicity and high level of religiosity were significantly associated with the “Interpersonal comfort” domain (adjusted odds ratio (95% confidence interval) (aOR (95% CI)) = 0.26 (0.09–0.78) and 0.34 (0.13–0.85), respectively). High level of religiosity was positively associated with the “Sex/gender beliefs” score (β = 0.11; 95% CI: 0.03–0.33). Female sex was positively associated with a favorable “Human value” attitude toward transgender patients at 4.03 (1.36–13.69), but residents had a negative aOR of 0.19 (0.04–0.85). In conclusion, our study identified physician characteristics associated with a less favorable attitude toward transgender patients. They should be the focus of interventions aimed at improving these attitudes.</p>