Preparing students for the transition to graduate-level education with greater learning demands in a condensed time frame is a challenging process for health professions educators and incoming students. Prematriculation programs offer a solution for exposing students to the foundational sciences in preparation for the academic rigor of a doctoral program. This retrospective study assessed whether incoming students enrolled across 3 yr of a 2-day online anatomy workshop, offered in July and August before the start of their first semester resulted in improved anatomy knowledge. Whether this acquired knowledge translated to improved anatomy outcomes in the first semester of a Doctor of Physical Therapy program was also assessed, while also accounting for variables of gender, ethnicity, and grade-point average. Knowledge acquired during both days of the workshop resulted in statistically significant improvements in anatomy postquiz scores compared to the baseline prequiz (P < 0.001). Multivariate regression analyses demonstrated statistically significant relationships between the first-semester anatomy practical score and workshop participation (P = 0.04) as well as a predictive value of gender (P = 0.01). Evaluating a timing effect on the predictive value of the online anatomy workshop demonstrated statistically significant effects of the prematriculation workshop on both first-semester anatomy practicals for August (P = 0.03 for practical 1; P = 0.04 for practical 2) but not July workshop participants. Findings from this study support the utility of an online prematriculation anatomy workshop to prepare students for graduate-level anatomy learning expectations in a doctoral allied health program.NEW & NOTEWORTHY This paper presents the findings of a retrospective study examining the effectiveness of an online prematriculation anatomy workshop on knowledge acquisition and first-semester anatomy competency following the success of a previously offered peer-led onsite workshop. To our knowledge, this is the first report of an online prematriculation program that successfully introduces graduate-level learning expectations and access to anatomical resources leading to improved anatomy competency in an allied health professional program.