Background: Differential diagnosis between adenoma and hyperplasia in primary hyperparathyroidism (pHPT) remains a dilemma. The aim of this study was to assess differences in transcriptional genomic expression profiles between sporadic (nonfamilial) parathyroid hyperplasia (SPH), adenoma, and normal tissue. Methods: Parathyroid tissue from 12 patients with parathyroid adenoma, 3 with SPH, and 2 with normal glands was selected for analysis. Histopathology was reviewed in all cases, and all patients with adenomas presented normocalcemia for a minimum of 6 months after one gland resection. Hybridizations were performed in a microarray containing 19,968 human cDNA clones including contiguous replicates. Direct comparisons were performed with reverse labeling for every different pooled sample entity. Expression levels were analyzed using the SAM, SMA, LIMMA, Cluster, and PAM packages in the R environment for statistical computing. Results: There were significant statistical differences between SPH and adenomas. In the direct comparison, a total of 200 genes showed differential expression (P < 0.03): 61 genes were upregulated (> 1.65-fold increase) and 139 were downregulated (> 1.58-fold decrease) with a B value > 4.68 (99.08% probability of real differential expression). When SPH was compared to normal parathyroid tissue, 50 genes were differentially expressed: 42 were upregulated (> 1.89) and 8 were downregulated (> 1.7) with a B > 4.26 (98.6% probability of real differential expression). At least 17 genes were differentially expressed and able to discriminate SPH from adenoma or normal tissue. Upregulated genes were related to apoptosis inhibition, cell proliferation, transcriptional activity and cell adhesion, among other activities. Downregulated genes were mainly related to ion channel activity, lipopolysaccharides, prostaglandin-d synthase, and integral membrane proteins. Conclusions: Our data suggest that SPH and adenoma have a singular molecular signature that, theoretically, could be used for the differential diagnosis of these entities and normal parathyroid tissue.