Anaplastic thyroid cancer (ATC) is among the most aggressive and unresectable tumors, presenting a bad prognosis. A better comprehension of the functional and molecular mechanisms behind the aggressiveness of this cancer, as well as new biomarkers for aggressiveness, prognosis, and response to therapy are required. However, owing to their irresectability, ATC tissue is not always accessible. Here we describe the establishment and characterization of a new patient-derived cell line, obtained from an unresectable ATC through fine-needle aspiration cytology (FNAC). The morphology, expression of epithelial and thyroid markers, cytogenetic, mutational and gene expression profiles, doubling time, and drug-resistance profile of the new cell line, designated C3948, were investigated using several methodologies: immunostaining, karyotype analysis, comparative genomic hybridization (CGH), fluorescent in situ hybridization (FISH), next-generation sequencing (NGS), Sanger sequencing, gene expression microarrays, cell counting, and IC50 determination. Results indicate that C3948 cell line has a histological phenotype representative of original ATC cells and a completely aberrant karyotype with many chromosomal losses and gains; harbors mutated TP53, STK11, and DIS3L2 genes; presents a gene expression profile similar to C643 ATC commercial cell line, but with some unique alterations; has a doubling time similar to C643; and the IC50 profile for paclitaxel, doxorubicin, and cisplatin is similar to C643, although higher for cisplatin. These observations are consistent with a typical ATC cell profile, supporting C3948 cell line as a novel preclinical model, and FNAC as a useful approach to better study anaplastic thyroid cancer, including testing of new anticancer therapies.