Abstract Purpose Definitive radiation therapy is the mainstay of treatment for early stage laryngeal squamous cell carcinoma (LSCC). However, up to 30% of the patients do not respond to radiotherapy. Unfortunately, we are unable to predict which tumors are likely to respond to radiation, and which will be resistant and persist. Therefore, the development of novel markers to predict response to radiotherapy is urgently needed. This study was designed to evaluate the expression pattern of microRNAs (miRNAs) in LSCC in order to identify markers capable of segregating radioresistant and radiosensitive tumors and to investigate the relationship between the expression of these miRNAs and the prognosis of LSCC. Methods The expression profile of 667 miRNAs was determined in an initial screening of nine early-stage LSCC samples (5 radioresistant and 4 radiosensitive) using TaqMan Low-Density Array (TLDA). Real-time polymerase chain reactions were performed to validate the expression of selected miRNAs in an expanded LSCC cohort (20 radioresistant and 14 radiosensitive). The miRNA expression level was scored as high or low based on the median of the expression in the LSCC samples. Results A comprehensive miRNA expression profiling enabled the identification of four miRNAs (miR-296-5p miR-452, miR-183* and miR-200c) differentially expressed in radioresistant LSCC. Moreover, the analysis of additional 34 LSCC samples, confirmed the expression of miR-296-5p as significantly related to radioresistance (p = 0.002) as well as an association of this marker with recurrence (p = 0.025) in early stage laryngeal cancer. Conclusions This study indicates that miR-296-5p expression is associated with resistance to radiotherapy and tumor recurrence in early stage LSCC, showing the feasibility of this marker as a novel prognostic factor for this malignance. Furthermore, miR-296-5p expression could be helpful in the identification of tumors resistant to radiotherapy; thus aiding the clinicians in the choice of the best therapeutic scheme to be used in each case.