Fine-needle aspiration biopsy (FNAB) is a widely employed diagnostic examination of the thyroid gland, especially important in a preoperative management of thyroid tumours. The molecular analysis of material collected from thyroid aspirates might represent a useful tool for improvement of accuracy of traditional cytology-based diagnostics of thyroid diseases. Nowadays, FNAB is generally believed to be a crucial procedure in a preoperative selection of thyroid nodules in terms of a risk of malignancy. However, this widely-used technique has certain intrinsic limitations, related particularly to relatively high risk of specimen inadequacy for microscopic evaluation, and the following formulation of specific diagnosis. Thus, the significant number of FNAB results appears to be non-diagnostic. In order to improve a diagnostic accuracy of the cytological examination of the thyroid, other methods, such as a computer-aided morphological image analysis, different immunocytological staining methods or molecular analysis of collected samples are performed. Novel techniques, used for molecular biology experiments, e.g., real-time polymerase chain reaction (RT-PCR), represent such a sensitivity level that provides reliable results, even when FNAB smears are assessed as insufficient for cytological evaluation (scarce cellularity), however, still allowing for genetic analysis of biological specimens, in spite of a small number of cells. In this review, we present a new panel of FNAB-based diagnostic tools for adequate analysis of thyroid disorders at the molecular level, facilitating a precise defining preoperative diagnosis of malignancy.