Male gonadal dysfunction is a frequent late effect after pediatric hematopoietic stem cell transplantation (HSCT), but detailed insights into patterns of male gonadal function at long-term is limited since studies have been retrospective without semen sample data. We investigated 1) the risk of azoospermia and testosterone deficiency, 2) the diagnostic value of markers of spermatogenesis, and 3) paternity at long-term follow-up after pediatric allogeneic HSCT. All surviving men ≥18 years of age transplanted in Denmark or Finland between 1980-2010 were invited to this cross-sectional study. Examinations included a semen sample, reproductive hormones, testicular volumes and screening for chronic graft-versus-host-disease (GvHD). Cumulative (pre-HSCT plus HSCT) treatment doses were calculated. Of 181 eligible patients, 98 participated median (range) 18 (8-35) years after HSCT. Sperm was found in 30 patients, azoospermia in 42, and azoospermia during testosterone substitution in 24 patients. A higher cumulative testicular irradiation dose was associated with increased risk of azoospermia and testosterone substitution (odds ratio [OR] per +1 Gy 1.27, 95%CI 1.14-1.46); p<0.001 and 1.21, 95%CI 1.11-1.38; p<0.001, respectively). All patients treated with >12 Gy had azoospermia and all but one treated with >16 Gy needed testosterone substitution. In patients treated with chemotherapy only (n=23), a higher cumulative cyclophosphamide equivalent dose was associated with increased the risk of azoospermia (OR per +1 g/m2 1.34, 95%CI 1.01-2.15; p=0.037). Pre-pubertal stage at HSCT was a risk factor for testosterone substitution (OR 15.31, 95%CI 2.39-315; p=0.017), while chronic GvHD was unrelated to gonadal dysfunction. Inhibin B was the best surrogate marker of azoospermia (area under the curve 0.91, 95%CI 0.85-0.98, 90% sensitivity and 83% specificity) compared to follicle-stimulating hormone and testicular volume. Of 24 males who had attempted to conceive, six had fathered children. In conclusion, the risk of male gonadal dysfunction after pediatric HSCT is high and depends primarily on the cumulative testicular irradiation dose and pubertal stage at transplant. Our findings pinpoint the need of fertility preservation before HSCT as well as prolonged follow-up of pediatric HSCT patients into adulthood.