Objective To investigate the timing and features of fine-needle aspiration biopsy(FNAB)guided by ultrasound for Bethesda Ⅲ thyroid nodules and to further optimize the puncture scheme.Methods A retrospective analysis was conducted on data from patients who underwent rFNAB for Bethesda Ⅲ thyroid nodules at the China-japan Union Hospital of Jilin University Thyroid Surgery Department from Dec.2020 to Dec.2022.The study included 71 cases(73 nodules),consisting of 57 females and 14 males,with an average age of(45.7±10.4)years.Patients were grouped based on rFNAB results:Bethesda Ⅱ as the benign group(n=21),Bethesda Ⅴ/Ⅵ as the malignant group(n=39),and the remaining cases categorized as the indeterminate group(n=13).Data on rFNAB results,puncture interval time,postoperative pathology,ultrasound features,and FNAB characteristics were recorded.Descriptive statistics were used for categorical data,presented as percentages and numbers,while contin-uous data were presented as mean±standard deviation((x)±s).The x2 test or Fisher's exact test was applied for analysis.Results In this study,rFNAB was performed on 73 Bethesda Ⅲ thyroid nodules.The results showed that 60 nodules(82.2%)received a definite diagnosis,while 13 nodules(17.8%)were indeterminate.There was a sta-tistically significant difference in the calcification classification between the malignant group and the benign group as well as the indeterminate group(P<0.05).Among the malignant group,29 patients underwent surgical treat-ment,with only 2 cases(8.0%)showing intermediate recurrence risk when surgery was performed more than three months after the initial FNAB.Upon reevaluation of the 31 initial FNAB samples from the malignant group,9 sam-ples(29.0%)exhibited mild cytological atypia,and 22 samples(71.0%)had poor quality specimens,mainly com-prising fibrous and calcified components.Conclusions For nodules initially classified as Bethesda Ⅲ,if suspi-cious ultrasound features persist or emerge,especially if microcalcifications are present,rFNAB should be per-formed after a follow-up period exceeding three months.However,during the puncture,multiple points should be targeted at the non-calcified areas of the nodule to enhance the accuracy and reliability of rFNAB.