This study investigates the expression of microRNAs (miRNAs) in 93 primary human breast tumors using a bead-based flow cytometric miRNA expression profiling method. Among the 309 human miRNAs assayed, 133 were found to be expressed in human breast and breast tumors. The expression of these miRNAs was associated with the molecular subtypes of breast tumors, including luminal A, luminal B, basal-like, HER2+, and normal-like. Several miRNAs were differentially expressed between these subtypes and were correlated with clinicopathological factors. The study also found that miRNA expression could classify basal versus luminal tumor subtypes in an independent dataset. Changes in miRNA expression were not fully explained by genomic loss or gain, but were likely due to changes in primary transcription or miRNA biogenesis. The expression of DICER1 and AGO2 was correlated with tumor subtype and may contribute to the observed changes in miRNA expression. This integrated analysis of miRNA expression, mRNA expression, and genomic changes in human breast cancer provides a basis for functional studies of miRNAs in breast cancer etiology and suggests that bead-based flow cytometric miRNA expression profiling could be a suitable platform for classifying breast cancer into prognostic molecular subtypes.This study investigates the expression of microRNAs (miRNAs) in 93 primary human breast tumors using a bead-based flow cytometric miRNA expression profiling method. Among the 309 human miRNAs assayed, 133 were found to be expressed in human breast and breast tumors. The expression of these miRNAs was associated with the molecular subtypes of breast tumors, including luminal A, luminal B, basal-like, HER2+, and normal-like. Several miRNAs were differentially expressed between these subtypes and were correlated with clinicopathological factors. The study also found that miRNA expression could classify basal versus luminal tumor subtypes in an independent dataset. Changes in miRNA expression were not fully explained by genomic loss or gain, but were likely due to changes in primary transcription or miRNA biogenesis. The expression of DICER1 and AGO2 was correlated with tumor subtype and may contribute to the observed changes in miRNA expression. This integrated analysis of miRNA expression, mRNA expression, and genomic changes in human breast cancer provides a basis for functional studies of miRNAs in breast cancer etiology and suggests that bead-based flow cytometric miRNA expression profiling could be a suitable platform for classifying breast cancer into prognostic molecular subtypes.