Anatomy is a key knowledge area in chiropractic and is formally offered in the undergraduate component of chiropractic education. There is the potential for loss of anatomy knowledge before the opportunity to apply it in a clinical setting. This study aimed to determine whether chiropractic clinicians retain a level of anatomy knowledge comparable to that of chiropractic students and to compare chiropractors' self-rating of their anatomical knowledge against an objective knowledge assessment tool. A previously validated multiple-choice test was utilized to measure retention of limb musculoskeletal (MSK) knowledge in Australian chiropractors. One hundred and one registered chiropractors completed the questionnaire and responses were scored, analyzed, and compared to scores attained by undergraduate and postgraduate chiropractic students who had previously completed the same questionnaire. The results indicated that practitioners retained their anatomy knowledge, with a significantly higher total mean score than the undergraduate group [total mean score = 36.5% (±SD 13.6%); P < 0.01] but not significantly different to the postgraduate group [total mean score = 52.2% (±SD 14.1%); P = 0.74]. There was a weak positive correlation between chiropractors' self-rated knowledge and test performance scores indicating the effectiveness of this Australian chiropractic group in self-assessing their anatomy knowledge. This study found that Australian chiropractors' knowledge of MSK anatomy was retained during the transition from university to clinical practice and they accurately evaluated their own test performance.