Abstract Background Main challenges in thyroid cancer management are low sensitivity of preoperative differential diagnostics and absence of sufficient prognostic markers for choosing of therapeutic strategy, including decision regarding radioiodine therapy. The study aims evaluation of clinical utility of analyses of comprehensive set of driver and secondary mutations with targeted high-throughput sequencing. Methods Point mutations in 52 genes, CNVs at 1q, 9q, 17q, 22q, and fusions of 66 genes were analyzed with targeted high-throughput sequencing. The analyzed mutations included well known driving and putatively driving mutations selected from TCGA project and literature, and secondary mutations with known or putative prognostic significance. Enrichment of regions of interest was performed with AmpliSeq technology. Fusions were detected by the presence of chimeric transcripts. Sequencing was performed on Illumina NextSeq (for point mutations and CNVs) or MiSeq (for fusions). Samples included 64 confirmed cancer samples (41 samples with Bethesda IV-V cytology, and 23 samples - Bethesda VI cytology), 16 confirmed benign samples, 12 normal tissue samples. Results Mutations detected in carcinomas included, in addition to BRAF and RASs: CDKN2B, ZFHX3, PDGFRA, KIT, NTRK3, ECM2, TERT, KDR, KMT2A, TP53, RNF213, APC, CHEK2, PTEN, PPM1D, ATR, CDKN2A, CTNNB1, ARID1B, KMT2C, ALK, DICER1, fusions of NTRK3, NTRK1, PPARG and RET, and CNVs 22q-del и 9q-del. The detected mutations allow to characterize all studied cancer cases. In benign nodules mutations in NRAS, TSC2, KDR, EIF1AX, ARID1B, TSHR, ZFHX3, GRIN3A, DICER1, HRAS, CHEK2 were found. Conclusions Tests limited to BRAF, K/N/HRAS, RET-PTC1/3, PAX-PPARG mutations have low diagnostic value. The developed panel allows analyzing an expanded range of mutations, including recently described rearrangements and CNV. Design of the panel, initial validation and sequencing of cancer samples were funded by Foundation for Assistance to Small Innovative Enterprises in Science and Technology (№ 442ГС2/9119 - С3-40846); sequencing of benign samples was supported by the grant of the Russian Science Foundation (Agreement 19-75-00053). Legal entity responsible for the study The authors. Funding Foundation for Assistance to Small Innovative Enterprises in Science and Technology (442ГC2/9119 - C3-40846); Russian Science Foundation (Agreement 19-75-00053). Disclosure All authors have declared no conflicts of interest.