BACKGROUND. Analysis of different tumor markers by reverse transcriptase‒polymerase chain reaction (RT‐PCR) in fine‐needle aspiration samples of thyroid nodules has been studied with the objective of improving the accuracy of the preoperative diagnosis of thyroid lesions. The aim of the current study was to investigate thyroid fine‐needle aspiration samples inadequate for RT‐PCR analysis and to determine whether there is a correlation between their proportion and the method of sampling used or the greatest dimension of the nodules. METHODS. A total of 350 fine‐needle aspiration samples from patients with thyroid nodules were analyzed. After the aspirate was smeared for conventional cytology, the leftover material in the needle was used for RT‐PCR analysis in 1 group of 175 patients. In another group of 175 patients, a separate puncture was performed to obtain material for RT‐PCR analysis only. Samples were considered adequate for RT‐PCR analysis if the expression of both glyceraldehyde‐3‐phosphate dehydrogenase and thyroglobulin was found by RT‐PCR. RESULTS. In total, 61 (17.4%) samples inadequate for RT‐PCR were detected. All 12 samples that were inadequate for cytologic diagnosis were also found to be inadequate for RT‐PCR analysis. The proportion of inadequate samples for RT‐PCR was found to be significantly higher in samples taken from leftover material in the needle (21.7%) then in samples from a separate puncture (13.1%) (P = .049). No statistically significant correlation between the adequacy of samples for RT‐PCR and the largest dimension of the nodule was found. CONCLUSIONS. The proportion of samples inadequate for RT‐PCR was found to be higher in samples taken from leftover material in the needle than in samples obtained from a separate puncture. Cancer (Cancer Cytopathol) 2008. © 2008 American Cancer Society. Samples inadequate for reverse transcriptase‒polymerase chain reaction (RT‐PCR) analysis were studied from 350 thyroid fine‐needle aspiration samples. The proportion of inadequate samples for RT‐PCR was higher in samples taken from leftover material in the needle (21.7%) then in samples obtained from a separate puncture (13.1%) (P = .049), and no correlation was noted between the adequacy of samples for RT‐PCR and the largest dimension of the nodule.