SummaryBackground  Distinguishing malignant thyroid nodules in patients with follicular cytology by fine‐needle aspiration (FNA) remains problematic. The large majority of thyroid nodules (> 85%) are overtreated. Therefore, a clear need exists to develop more accurate initial diagnostic tests for follicular thyroid nodules. Galectin‐3 is the most recent promising marker to aid discrimination between benign and malignant thyroid lesions; however, this biomarker can be absent in follicular malignancies.Aims  This study was undertaken to determine whether additional biomarkers can help to discriminate between benign and malignant thyroid nodules.Methods  Surgical specimens of 36 patients with benign (n = 12) and malignant (n = 24) thyroid nodules showing follicular cytology were assessed by immunohistochemistry for the expression of galectin‐3 and novel biomarkers.Results  Expression of hexokinase III (HK III) (P = 0·000) cyclin A (P = 0·002) and galectin‐3 (P = 0·003) differed significantly between benign and malignant thyroid nodules. HK III had a sensitivity of 79% [95% confidence interval (CI) 60–91] and a specificity of 100% (95% CI 76–100) in predicting malignancy. Galectin‐3 had a sensitivity of 79% (95% CI 56–91) and a specificity of 75% (95% CI 47–91) in predicting malignancy. Combining HK III, cyclin A and galectin‐3 in a parallel test increased the sensitivity to 96% (95% CI 80–99) while the specificity remained at a high level of 75% (95% CI 47–91). Leave‐one‐out cross‐validation demonstrated a stable predictive validity of a model based on HK III, cyclin A and galectin‐3.Conclusions  In this study, we have demonstrated that in addition to galectin‐3, HK III and cyclin A profiles could be important biomarkers in predicting malignancy in follicular thyroid nodules. The use of these biomarkers may allow an accurate preoperative diagnosis of thyroid cancer, which can be cost saving and may avoid serious morbidity such as vocal cord paralysis. The value of the suggested biomarkers warrants further evaluation in a large prospective study on cytological samples of follicular thyroid nodules.