The event of X chromosome inactivation induced by , which is physiologically observed in females, is retained in testicular germ cell tumors (TGCTs), as a result of a supernumerary X chromosome constitution. X chromosome inactivation also occurs in male germline, specifically during spermatogenesis. We aimed to analyze the promoter methylation status of in a series of TGCT tissues, representative cell lines, and testicular parenchyma.Two independent cohorts were included, comprising a total of 413 TGCT samples, four (T)GCT cell lines, and 86 testicular parenchyma samples. The relative amount of methylated and demethylated promoter fragments was assessed by quantitative methylation-specific PCR (qMSP) and more sensitive high-resolution melting (HRM) methylation analyses.Seminomas showed a lower amount of methylated fragments as compared to non-seminomas or normal testis ( < 0.0001), allowing for a good discrimination among these groups (area under the curve 0.83 and 0.81, respectively). Seminomas showed a significantly higher content of demethylated as compared to non-seminomas. The percentage of demethylated fragment in cell lines reflected their chromosomal constitution (number of extra X chromosomes). A novel and strong positive correlation between the Johnsen's score and demethylation was identified (r = 0.75, < 0.0001).The X chromosome inactivation event and demethylated promoter are promising biomarkers for TGCTs and for assessing spermatogenesis quality.