The aim of this study was to develop a practical technique to detect mRNA expression and to validate a panel of mRNA markers for molecular differential diagnosis of renal cell carcinoma (RCC). The renal cancer cell line SKRC-52 was used to set up the technique, which consisted of column extraction of RNA and one-step reverse transcription-PCR. We validated a panel of gene markers, including MN/CA9, cadherin-6, vimentin, mucin1, and parvalbumin, and studied 50 renal tumors (30 conventional, 9 papillary, and 5 chromophobe RCCs and 6 oncocytomas), 10 normal tissues, and 10 normal blood samples. We mimicked fine needle aspiration (FNA) biopsy in 10 kidneys with conventional RCC and applied this technique to 10 preoperative FNA samples from imaging-indeterminate renal tumors. The technique could detect as few as 10 SKRC-52 cells with MN/CA9 as mRNA marker and was less time consuming and labor intensive. MN/CA9 was a sensitive and rather specific gene marker for conventional RCC. Cadherin-6 gene expression was a sensitive marker for conventional and papillary RCC. Vimentin was highly specific for conventional RCC. Mucin1 mRNA was sensitive for papillary and chromophobe RCC and oncocytoma. Parvalbumin mRNA was a sensitive and highly specific marker for both chromophobe RCC and oncocytoma. Thus, these mRNA markers represent the biomarker genes for the subtypes of renal tumors. Finally, we successfully applied the technique to FNA specimens. Five preoperative FNA samples were MN/CA9 gene positive, suggesting a RCC, whereas the routine cytology was positive in only three cases. A rapid and sensitive assay of mRNA markers was developed for molecular differential diagnosis of RCC. This molecular assay can be used as a powerful ancillary to surgical pathological diagnosis and cytological diagnosis of RCC.