Testicular Adrenal Rest Tumors (TART) are a common complication in patients with Congenital Adrenal Hyperplasia (CAH), leading to irreversible damage of the testes and infertility. The origin and etiological features of these benign tumors are unclear. It was hypothesized that TART is derived in utero from adrenal rests that fail to regress due to the elevated adrenocorticotropin hormone (ACTH) levels in CAH patients. However, next to adrenal characteristics, also testicular characteristics have been reported for TART, suggesting a more pluripotent progenitor as the origin of TART. This study aims to characterize TART using an unbiased approach, by comparing transcriptomic profiles from 14 TART with 11 adult adrenal-, 10 adult testis-, 13 fetal adrenal -and 5 fetal testis- tissues. Knowledge on the origin and etiological pathways of TART will aid in the development of therapies to inhibit the growth of TART. The origin of TART is explored using clustering analysis. TART-specific functional characteristics and pathways were identified using differential expression analysis, followed by Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Clustering analysis corroborates the similarity of TART with adult adrenal tissue. Functional annotation and pathway analysis of 507 genes significantly upregulated in TART compared to normal adult testis (LogFC > 2; adjusted p-value < 0.05) pointed out enriched GO terms and pathways associated with steroid metabolism and receptor binding. Analysis of 830 genes upregulated in TART versus adult adrenal tissue highlighted molecular functions and biological processes involved in extracellular matrix (ECM) organization / remodeling and angiogenesis. Six KEGG pathways were enriched in TART compared to adrenal tissue, of which three are known to be involved in fibrosis. Further studies should focus on the analyses and validation of those TART-specific characteristics and pathways as they may comprise therapeutic targets to inhibit the progression of TART.