Background: MicroRNA (miRNA) are short noncoding RNAs involved in regulation of gene expression and have shown promise as novel biomarkers in the diagnosis and management of testicular germ cell tumors (GCT). We report the accuracy of our combined miRNA assay in men with Clinical Stage I (CSI) GCTs. We also sought to explore the relationship between miRNA levels and patient and oncologic variables to better understand how these factors may influence miRNA results. Methods: We retrospectively identified patients at Memorial Sloan Kettering (MSK) with a newly identified testicular mass concerning for GCT who were planned for radical or partial orchiectomy and had pre-orchiectomy MSK miRNA Assay (miRNA-371a-3p and miRNA-372-3p, collectively MMA) drawn. All patients had clinically localized disease based on initial staging imaging and normalized traditional serum tumor markers (STM) post-orchiectomy. Specific miRNA levels were determined using stem-loop RT-qPCR (quantitative reverse transcription PCR), as described previously. Patient and disease factors were abstracted from the medical record. Outcomes of interest were sensitivity, specificity, PPV and NPV of MMA, and area under the receiver operating characteristic curve (AUC) of miRNA-371a-3p and 372-3p levels, calculated relative to detection of viable GCT at orchiectomy. We explored the relationship between pre-orchiectomy miRNA-371a-3p and 372-3p levels and various patient and pathologic factors using Spearman correlation and Wilcoxon rank-sum test. Results: Forty-eight patients were included in this study, 39 of whom had viable GCT at orchiectomy and 9 with no cancer or non-GCTs. Thirty-seven patients with GCT had positive pre-orchiectomy MMA results (sensitivity 94.9%, 95% CI: 88-100), while 1 of 9 patients (Leydig cell tumor) without viable GCT had a positive MMA result (specificity 88.9%, 95% CI: 68.4-100). MMA PPV was 97.4% (95% CI: 92.3-100) and NPV was 80% (95% CI: 55.2-100). AUC of miRNA-371a-3p and miRNA-372-3p was 96% (95% CI: 91.0-100) and 92% (95% CI: 84.2-99.9), respectively. Pre-orchiectomy miRNA-371a-3p and miRNA-372-3p levels were positively correlated with tumor size (Spearman coefficient ρ=0.64, 0.63, respectively; p<0.001), and with serum HCG (ρ=0.55, 0.56; p<0.001) and LDH (ρ=0.46, 0.46; p<0.001), but not AFP (ρ=0.19, 0.17; p=0.20, 0.24). Pre-orchiectomy MMA was not associated with other tested pathologic or patient characteristics including age, BMI, renal function, liver function and andrology labs. Conclusions: miRNA 371a-3p and 372-3p are highly sensitive and specific for presence of viable GCT in patients with clinically localized disease. Tumor size was the only studied pathologic factor or patient characteristic correlated with MMA levels.