<jats:sec><jats:title>Background</jats:title><jats:p>Telomeres are tandem repeats of TTAGGG at the end of eukaryotic chromosomes that play a key role in preventing chromosomal instability. The aim of the present study is to determine telomere length using fluorescence in situ hybridisation (FISH) on cytological specimens.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>Aspiration samples (n = 41) were smeared on glass slides and used for FISH.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Telomere signal intensity was significantly lower in positive cases (cases with malignancy, n = 25) as compared to negative cases (cases without malignancy, n = 16), and the same was observed for centromere intensity. The difference in DAPI intensity was not statistically significant. The ratio of telomere to centromere intensity did not show a significant difference between positive and negative cases. There was no statistical difference in the signal intensities of aspiration samples from ascites or pleural effusion (n = 23) and endoscopic ultrasound‐guided FNA samples from the pancreas (n = 18).</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>The present study revealed that telomere length can be used as an indicator to distinguish malignant and benign cells in cytological specimens. This novel approach may help improve diagnosis for cancer patients.</jats:p></jats:sec>