Cardiovascular diseases are the leading cause of death globally, with the limited regenerative ability of the adult heart hindering recovery after injury. Telomere dysfunction, associated with decreased telomerase (TERT/TERC) activity, leads to cardiomyocyte cell cycle exit and cardiac failure, while cardiac progenitor cells like Sca-1+ cells offer a potential source for regeneration. This study investigated the effect of TERT deficiency on Sca-1+ progenitor cells using Q-FISH and CO-FISH analyses. No significant differences were found between TERT-/- and TERT+/+ cells in terms of average telomere length, telomere distribution, or telomeric sister chromatid exchange. However, TERT-/- cells showed an increase in signal-free ends, indicating impaired maintenance of critically short telomeres and genomic instability. These findings suggest that although global telomere parameters are maintained, TERT is crucial for preserving telomere integrity in cardiac progenitor cells, with implications for heart regeneration.