Long-term testosterone replacement therapy is mainly monitored by trough levels of serum testosterone (S-T), while urinary testosterone (U-T) is used by forensic toxicology to evaluate testosterone doping. Testosterone in saliva (Sal-T) may provide additional information and simplify the sample collection. We aimed to investigate the relationships between testosterone measured in saliva, serum and urine during standard treatment with 1,000 mg testosterone undecanoate (TU) every 12th week during 1 year. This was an observational study. Males with primary and secondary hypogonadism (HG; n = 23), subjects with gender dysphoria (GD FtM; n = 15) and a healthy control group of men (n = 32) were investigated. Sal-T, S-T and U-T were measured before and after TU injections. Sal-T was determined with Salimetrics enzyme immunoassay, S-T with Roche Elecsys testosterone II assay and U-T by gas chromatography-mass spectrometry. Sal-T correlated significantly with S-T and calculated free testosterone in both controls and patients (HG men and GD FtM), while Sal-T to U-T showed weaker correlations. Trough values of Sal-T after 12 months were significantly higher in the GD FtM group (0.77 ± 0.35 nmol/L) compared to HG men (0.53 ± 0.22 nmol/L) and controls (0.46 ± 0.15 nmol/L), while no differences between S-T and U-T trough values were found. Markedly elevated concentrations of salivary testosterone, 7-14 days after injection, were observed, especially in the GD FtM group. This study demonstrates that Sal-T might be a useful clinical tool to monitor long-term testosterone replacement therapy and might give additional information in forensic cases.