--- - |2- Abstract The relationship between the three‐dimensional (3D) nuclear telomere architecture and specific genetic alterations in papillary thyroid carcinoma (PTC), in particular in cancer stem‐like cells (CSLCs), has not yet been investigated. We isolated thyrospheres containing CSLCs from B‐CPAP, K1, and TPC‐1 PTC‐derived cell lines, representative of tumors with different genetic backgrounds within the newly identified BRAFV600E‐like PTC subgroup, and used immortalized normal human thyrocytes (Nthy‐ori 3.1) as control. We performed quantitative fluorescence in situ hybridization, 3D imaging, and 3D telomere analysis using TeloView software to examine telomere dysfunction in both parental and thyrosphere cells. Among the 3D telomere profile, a wide heterogeneity was observed, except for telomere intensity. Our findings indicate that CSLCs of each cell line had longer telomeres than parental cells, according to telomere intensity values, which correlate with telomere length. Indeed, the thyrosphere cells had lower numbers of lower‐intensity telomeres (≤5,000 arbitrary fluorescent units, a.u.), compared with parental cancer cells, as well as parental control cells, (p < 0.0001). The B‐CPAP thyrospheres showed a decreased number of higher intensity telomeres (>17,000 a.u.) than K1 and TPC‐1 cells, as well as control cells (p < 0.0001). By selecting PTC‐derived cell lines with different genetic backgrounds characteristic of BRAFV600E‐like PTC subgroups, we demonstrate that thyrosphere cells with BRAFV600E and TP53 mutations show shorter telomeres than those harboring RET/PTC or BRAFV600Eand wild‐type TP53. Hence, our data reveal a trend towards a decrease in telomere shortening in CSLCs, representing the early cancer‐promoting subpopulation, as opposed to parental cells representing the tumor bulk cells. - Journal of Cellular Physiology, EarlyView.