Purpose <p>Chronic autoimmune thyroiditis shows increased stiffness on elastography. The sternocleidomastoid (SCM) or strap muscles have been used as references in strain elastography in prior studies. We aimed to compare the effects of different reference points in strain elastography on its diagnostic performance.</p> Methods <p>Patients referred for thyroidectomy underwent strain elastography assessment of the non-tumor-bearing thyroid parenchyma. The strain ratio was determined using either the adjacent SCM muscle or the trachea at the same depth as reference points. Final pathological results served as the standard.</p> Results <p>A total of 174 patients were analyzed, with 39 (22%) diagnosed with autoimmune thyroiditis. Multivariate logistic regression identified thyroid peroxidase antibodies, thyroglobulin antibodies, and the strain ratio using the trachea as a reference as independent predictors of thyroiditis. The area under the receiver operating characteristic curve was significantly higher with the trachea as the reference compared to the SCM (0.854 vs. 0.611). Sensitivity analyses indicated that diagnostic performance was not affected by patient age or body habitus.</p> Conclusions <p>Different reference points in strain elastography significantly influence the predictability of autoimmune thyroiditis, and using the trachea as a reference improves diagnostic performance.</p>