Objective: A class of small non-coding RNAs (microRNAs) has been recently identified as critical molecules involved in a wide spectrum of normal and pathological processes. MicroRNAs (miRNAs) are negative regulators of gene expression and function as cytoplasmic gene silencers by suppressing translation of targeted mRNAs. They have a tumor specific profile which is variable in different tumor grades and stages. In this study, the expression of miR-21 (an oncomir upregulated in a variety of tumors) and miR-302 (an embryonic stem cells-specific miRNA, expressed in some cancer stem cells) was evaluated in formalin-fixed paraffin-embedded (FFPE) samples of esophagus cancers. FFPE archival tissue samples are a valuable source of materials for determining the retrospective prognostic value of miRNA profiling in human cancers. Materials and Methods: Tumor and non-tumor (adjacent, apparently-normal tissue of the same patients) samples of esophagus squamous cell carcinoma (SCC) were collected from the archival collection of Shiraz university of medical sciences. The tumor parts of the whole blocks were carefully punched off to avoid contamination with non-tumor parts of the blocks. Deparaffinization procedure as well as proteinase K digestion process was optimized using different strategies and solutions. Then, total RNA extraction was performed using Trizol and RNX solutions. cDNA synthesis and real-time RT-PCR were performed using Exiqon uniRT cDNA synthesis kit and SYBR green master mix. MiRCURY LNA™ microRNA detection probes were employed to localize subcellular distribution of miR-21, and miR-302 by means of in-situ hybridization (ISH). The ISH procedure was optimized by using different Proteinase K solutions and hybridization temperatures on an internal control (U6 snRNA). Results: We detected miR-21 and miR-302 expression in several stem and tumor cell lines, including KYSE30 cell line of esophagus. We also optimized a practical technique for total RNA extraction from FFPE samples and detected miR-21 and miR-302 overexpression in tumor samples compared to their non-tumor counterparts. We also detected the nuclear localization of U6 snRNA, as a control non-coding RNA, using ISH technique and by means of LNA probes. Conclusion: We were able to extract and amplify miRNAs from FFPE tumor samples. The obtained data are of great clinical importance and have potential usefulness to introduce new tumor markers capable of predicting the malignant behavior of different types of tumors.