Purpose: This study aimed to compare thyroid uptake values according to background measurement site (mid-thigh versus neck using a B-type filter) in ^99mTc-pertechnetate thyroid uptake studies, evaluate the agreement between the two methods, and assess the influence of sex-related differences. Materials and Methods: A retrospective analysis was performed in 98 patients who underwent ^99mTc-pertechnetate thyroid uptake studies. Background activity was measured at both the neck and mid-thigh under identical geometric conditions. Thyroid uptake values were calculated using each background method. Paired t-tests, Pearson correlation analysis, and Bland–Altman analysis were performed to compare the two methods. Sex-based differences in thyroid uptake values (thigh − neck) were additionally evaluated using an independent samples t-test. Results: Neck background activity measured using a B-type filter was significantly higher than mid-thigh background activity (21,472.2±4,268.8 cpm vs. 18,660.3±3,587.4 cpm, P < 0.001). Thyroid uptake calculated using thigh background was also significantly higher than uptake calculated using neck background (3.96±2.76% vs. 3.74±2.73%, P < 0.001). Thyroid uptake values obtained using the two background measurement methods were correlated (r = 0.994, P < 0.001). Bland–Altman analysis demonstrated a mean bias of 0.223%, with 95% limits of agreement ranging from −0.358% to 0.804%. Female patients showed a tendency toward a larger difference in thyroid uptake values than male patients, although the difference was not statistically significant (P = 0.121). Conclusion: Although neck background measurement using a B-type filter resulted in significantly higher background counts and slightly different thyroid uptake values compared with mid-thigh measurement, the two methods demonstrated overall agreement. Nevertheless, mid-thigh background measurement may provide a more stable assessment by reducing the influence of adjacent thyroid-related structures. These findings suggest that mid-thigh background measurement may be useful for more consistent thyroid uptake evaluation in ^99mTc-pertechnetate studies.