Global micro-RNA (miR) profiling of human malignancies is increasingly performed, but to date, the majority of such analyses have used frozen tissues. However, formalin fixation is the standard and routine histological practice for optimal preservation of cellular morphology. To determine whether miR analysis of formalin-fixed tissues is feasible, quantitative real-time PCR (qRT-PCR) profiling of miR expression in 40 archival formalin-fixed paraffin-embedded (FFPE) breast lumpectomy specimens were performed. Taqman Low Density Arrays (TLDAs) were used to assess the expression level of 365 miRs in 34 invasive ductal carcinomas and in 6 normal comparators derived from reduction mammoplasties. Its technical reproducibility was high, with intra-sample correlations above 0.9 and with 92.8% accuracy in differential expression comparisons, indicating such global profiling studies to be technically and biologically robust. The TLDA data were confirmed using conventional single-well qRT-PCR analysis, showing a strong and statistically significant concordance between these two methods. Paired frozen and FFPE breast cancer samples from the same patients showed a similar level of robust correlation of at least 0.94. Compared with normal breast samples, a panel of miRs was consistently dysregulated in breast cancer, including earlier-reported breast cancer-related miRs, such as upregulated miR-21, miR-155, miR-191, and miR-196a, and downregulated miR-125b and miR-221. Additional novel miR sequences of potential biological relevance were also uncovered. These results show the validity and utility of conducting global miR profiling using FFPE samples, thereby offering enormous opportunities to evaluate archival banks of such materials, linked to clinical databases, to rapidly acquire greater insight into the clinically relevant role for miRs in human malignancies.