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Background: Well characterized human cell lines are needed for preclinical treatment studies of anaplastic thyroid cancer (ATC). Aims/Objectives: The aim was to establish, verify and characterize a panel of ATC cell lines. Material and methods: Cell lines were established from ATC fine-needle aspiration biopsies and characterized genetically and functionally regarding treatment sensitivities. Results: Eight cell lines were established in vitro and the anaplastic thyroid origin was verified. Seven of the cell lines were also grown as xenografts. The cell lines harboured complex karyotypes with modal numbers in hyperdiploid to near-pentaploid range. Five were TP53 mutated and three carried the BRAF V600E mutation. None had rearrangements of RET . For doxorubicin, IC 50 ranged from 0.42 to 46 nmol/L and for paclitaxel from 1.6 to 196 nmol/L. Radiation sensitivity varied between 2.6 and 6.3 Gy. Two of the BRAF mutated cell lines displayed high sensitivity to vemurafenib, while the third was similar to the wild-type ones. Conclusions and significance: We describe a series of new ATC cell lines demonstrating large heterogeneity in the response to cytostatic drugs and the BRAF inhibitor vemurafenib. The observations are relevant to future attempts to optimize treatment combinations for ATC.