The profession of physical therapy has undergone an evolution since its inception. Since the early 1900s it has grown from a technical training program to a doctorate level degree. Human anatomy courses remain a requirement for physical therapist educational curricula. However, changes in anatomy pedagogy have been trending within health profession educational models, leading to questions regarding which method is best for student learning. The objective of this study was to determine if anatomy instructional method used within physical therapist educational curricula impacted current anatomy knowledge. Licensed physical therapists were recruited to complete a demographic survey and a questionnaire to demonstrate their knowledge of anatomy topics. Anatomy topics included six regional components: (1) upper limb; (2) lower limb; (3) thorax and abdomen; (4) pelvis; (5) spine; and (6) head. Each regional component contained five questions regarding systemic subsets related to joints and osteology, muscles, nervous system, vasculature, and special areas (e.g., spatial orientations, structures within spaces, pathways of nerves). Within the thorax and abdominal region, data analysis indicated that the dissection instruction method, when compared to no laboratory instruction, led to statistically significant greater anatomical knowledge (P = 0.02). Dissection also showed greater means when compared to the no laboratory method (P = 0.02) and the prosection method in the head region (P = 0.01). However, the variance explained by instructional method was small. This study adds empirical evidence regarding current anatomy knowledge exhibited by physical therapists as the level of anatomical knowledge exhibited small differences based on instructional methods.