Human brain is a complex organ comprising multiple cell types of differing function. Although histological evaluation remains the mainstay approach for evaluating tissue, comprehensive molecular characterization is now possible due to advanced -omic approaches. microRNAs (miRNAs) are small (~22 nt) RNA molecules that regulate gene expression and mediate cellular differentiation in normal brain development. miRNAs also make excellent tissue markers due to their abundance, cell-type and disease-stage specificity, and stability in solid/liquid clinical samples. To advance our knowledge of miRNA-mediated gene regulation in human brain, we generated comprehensive miRNA expression profiles from 117 fresh normal brain samples through barcoded small RNA sequencing; tissues included neocortex, allocortex, white matter, cerebellum, olfactory bulb, optic nerve, pineal gland and spinal cord. FASTQ sequence files were annotated using state-of-the-art sequence annotation available through the Renwick lab. Following data pre-processing, high expression analysis of miRNA profiles showed that miR-9 was the highest expressed miRNA in neocortex, cerebellum and olfactory bulb, whereas miR-22 was highest expressed in cingulate cortex, optic nerve and spinal cord; interestingly, miR-29 was the highest expressed miRNA in hippocampus. Our analyses showed a trend towards unique miRNA signatures in different anatomical areas of the brain. Our next step is to perform miRNA fluorescence in situ hybridization on formalin-fixed paraffin-embedded tissues using a novel method developed in the Renwick lab. Accurate miRNA characterization of normal tissues will provide a firm basis for understanding miRNA changes in neurological diseases.