Hurthle cell tumours of thyroid are regarded as a subtype of follicular thyroid tumours and are a rare type of disease. Despite of recent large improvement in diagnostics of cancer patients, a definitive way to differentiate a benign tumour (Hurthle cell thyroid adenoma - HCTA and Hurthle cell thyroid nodule - HCTN) from Hurthle cell thyroid carcinoma (HCTC) is based on histological examination of thyroid tissue, where vascular and/or transcapsular invasion is characteristic for HCTC. For HCTA or HCTN, lobectomy is a sufficient surgical procedure. However, if HCTC is diagnosed by histopathology after lobectomy, then complete thyroidectomy is performed as a second surgical procedure. Use of appropriate genetic markers of HCTC, could help in early differentiation between benign tumour and cancer, as well as enable better treatment planning, including optimal surgical procedure (lobectomy or total thyroidectomy) in patients with HCTN. Hypothesis The following hypotheses were verified: a) Specific mutation of oncogenes and tumour suppressor genes are genetic markers of HCTC, disease recurrence, or distant metastasis; b) Specific polymorphisms of antioxidative genes or DNA repair genes are genetic markers of HCTC, disease recurrence, or distant metastasis. Patients and methods A retrospective study included 139 patients treated by thyroid surgery for Hurthle cell neoplasm. We isolated genomic DNA from tumour and normal thyroid tissue of formalin-fixed and paraffin-embedded samples. We have screened 10 representative tumour DNA samples for 739 mutations of 46 oncogenes and tumour suppresion genes, using second generation DNA sequencing. Selected mutations, that were detected as possible markers of HCTC, were verified on all 139 tumour DNA samples, using high sensitivity real time polymerase chain reaction. In patients harbouring specific mutations in their tumour DNA, DNA analysis was repeated in their normal thyroid tissue, to verify whether the mutation is somatic or germinal. DNA from normal thyroid tissue was also analyzed for common functional polymorphism of antioxidant genes (CAT, SOD2, GSTM1, GSTT1, GSTP1, GPX1) and DNA repair genes (OGG1, XRCC1, NBN, RAD51, XRCC3). Results Patients were diagnosed as follows: 53 had HCTC, 37 HCTA, 21 HCTN and 18 had multinodular goiter, or follicular thyroid adenoma, or lymphocytic thyroiditis. Altogether 20 of 53 patients with HCTC had metastatic disease. Recurrent disease was observed in 16 patients with HCTC. The patients from the HCTC group had different gender (F/M) ratio (p = 0.043), were older (p = 0.004) and had larger initial tumour diameter (p < 0.001) in comparison with the patients from the HCTA or HCTN group. By second generation DNA sequencing, 26 (of totally 46) mutated oncogenes and tumour suppression genes were found and were more often in HCTC groups. Mutations in selected genes (BRAF, KRAS, NRAS) were verified in all the remaining samples, but no associations between gene mutations and presence of HCTC and HCTA or HCTN groups were found. All the detected mutations in these genes were somatic. Under the dominant genetic model, no significant differences in the genotype frequency distribution of antioxidative genes and DNA repair genes were observed, when HCTC group was compared to HCTA and HCTN groups. These polymorphisms were also not associated with presence of metastatic disease. However, GPX1 polymorphism was associated with the presence of recurrent disease (p = 0.040). Conclusions In our study we did not find any association between selected mutations of BRAF, KRAS and NRAS and presence of HCTC, or metastatic, or recurrent disease. All detected mutations of these genes were somatic. In our study we also did not find any association between common functional polymorphism of antioxidant genes or DNA repair genes and development of HCTC, or metastatic disease. However, GPX1 polymorphism is associated with the risk of HCTC recurrence.