RNA transcript analysis is a promising method being used to better identify and understand allograft rejection, but its applicability to analyzing tolerance in heart transplantation has been limited by the lack of tolerant heart samples. Here, we utilized the Nanostring platform for RNA transcript analysis of a unique set of tissue samples from cynomolgus macaques tolerant of cotransplanted heart and kidney allografts. Total RNA was isolated from 144 formalin fixed and paraffin embedded (FFPE) nonhuman primate biopsy and autopsy samples and analyzed for mRNA expression of a custom panel of 216 genes with normalization against housekeeping genes. Differential expression analyses were performed on heart samples from tolerant animals, rejecting animals, animals on immunosuppression and naïve animals (native heart). Pathway analysis was performed using Gene Set Enrichment Analysis (GSEA). Tolerant heart allografts exhibited a signature with higher expression of cardiac genes, endothelial and angiogenesis related genes, T-cell regulatory genes, and stem cell related genes compared to rejecting organs while rejecting organs exhibited a signature with higher expression of B-cell and immunoglobulin genes, apoptosis related genes, and immune activation. In GSEA analysis, the most enriched pathway in rejecting animals was antigen stimulation through IgG and B-cells. Tolerant heart samples had similar gene expression profiles to immunosuppressed heart samples, both of which were markedly altered compared to naïve hearts. Nanostring analysis of mRNA expression in tolerant NHP cardiac allografts was performed for first time. RNA profiling could distinguish tolerant allografts from rejecting allografts. Surprisingly, the tolerance profile resembled that observed in allografts from stably immunosuppressed recipients instead of naïve hearts, possibly reflecting stable regulatory changes in indirect immunity established during immunosuppression that persist in tolerant animals but not in rejecting animals.