Despite a long history of using human donor dissection (HDD) for physical therapy (PT) anatomy education, there are no PT guidelines that require HDD. The purpose of this quantitative causal-comparative study was to determine if Doctor of Physical Therapy students who used HDD had different grades both within anatomy and within courses that require retention and application of anatomical knowledge (kinesiology and a foundational musculoskeletal course) compared with those who used virtual 3-dimensional anatomical software (VAS). Numerous factors affect the decision to use HDD within PT anatomy, and few PT studies have compared the effectiveness of VAS to HDD. All students who took anatomy in an entry-level PT program from 2018 to 2021 (232 total students, 115 who used HDD in 2018-2019 and 117 who used VAS in 2020-2021). Mann-Whitney tests were used to compare anatomy grades (course, written examination, and practical examination) and future grades in kinesiology and a foundational musculoskeletal course for students who used HDD or VAS. Physical therapy students who used VAS had statistically significant higher anatomy course grades (VAS 93.81% ± 4.99% to HDD 92.20% ± 4.53%) and higher practical examination grades (VAS 97.43% ± 2.91% to HDD 93.55% ± 4.39%) compared with those who used HDD. However, there were no significant differences between groups on written anatomy examinations (VAS 89.42% ± 7.21% to HDD 90.40% ± 4.94%), kinesiology grades (VAS 91.86% ± 4.52% to HDD 92.80% ± 4.27%), or foundational musculoskeletal grades (VAS 89.50% ± 3.89% to HDD 89.77% ± 3.83%). The causal-comparative study design prevents concluding that PT student grade differences were due exclusively to either anatomy laboratory method. It does provide preliminary evidence that the PT anatomy laboratory method did not practically affect anatomy performance or long-term application of anatomy knowledge in future coursework.