Formalin-fixed, paraffin-embedded tissues are an invaluable tool for biomarker discovery and validation. As these archived specimens are not always compatible with modern genomic techniques such as gene expression arrays, we assessed the use of microRNA (miRNA) as an alternative means for the reliable molecular characterization of formalin-fixed, paraffin-embedded tissues. Expression profiling using two different microarray platforms and multiple mouse and human formalin-fixed, paraffin-embedded tissue types resulted in the correlation ratios of miRNA expression levels between frozen and fixed tissue pairs ranging from 0.82 to 0.99, depending on the cellular heterogeneity of the tissue type. The same miRNAs were identified as differentially expressed between tissues using both fixed and frozen specimens. While formalin fixation time had only marginal effects on microarray performance, extended storage times for tissue blocks (up to 11 years) resulted in a gradual loss of detection of miRNAs expressed at low levels. Method reproducibility and accuracy were also evaluated in two different tissues stored for different lengths of time. The technical variation between full process replicates, including independent RNA isolation methods, was approximately 5%, and the correlation of expression levels between microarray and real-time quantitative reverse transcriptase polymerase chain reaction was 0.98. Together, these data demonstrate that miRNA expression profiling is an accurate and robust method for the molecular analysis of archived clinical specimens, potentially extending the use of miRNAs as new diagnostic, prognostic, and treatment response biomarkers.