Abstract Background Current cultural competence training needs were assessed as baseline measurement in Dutch physician assistant (PA) students and PA alumni that were not specifically trained in cultural competence. In particular, differences in cultural competency between PA students and PA alumni were assessed. Methods In this cross-sectional, observational cohort study knowledge, attitude, and skills and self-perceived overall cultural competence were assessed in Dutch PA students and alumni. Demographics, education and learning needs were collected. Total cultural competence domain scores as well as percentage of maximum scores were calculated. Results A total of 40 PA students and 96 alumni (female:75%; Dutch origin:97%) consented to participate. Cultural competence behavior was moderate in both groups. In contrast, general knowledge and exploration of patients’ social context were insufficient, i.e., 53% and 34%, respectively. Self-perceived cultural competence was significantly higher in PA alumni (6.5 ± 1.3, mean ± SD) than in students (6.0 ± 1.3; P < 0.05). Low heterogeneity among PA students and educator exists. Seventy percent of the respondents considers cultural competence important and the majority expressed a need for cultural competence training. Conclusions Dutch PA students and alumni have moderate overall cultural competence, but insufficient knowledge and exploring social context. Based on these outcomes the curriculum of the master of science program for physician assistant will be adapted.Emphasis should be made to increase the diversity of PA students to stimulate cross-cultural learning and developing a diverse PA workforce.