The aim of this work was to present the clinical and embryological outcomes of 65 azoospermic patients with non-mosaic Klinefelter syndrome (KS), treated by testicular sperm extraction (TESE), followed by intracytoplasmic sperm injection (ICSI), either with fresh or cryopreserved testicular spermatozoa. In total, spermatozoa were recovered in 25/65 (38.5%) of the cases. Of the 48 patients who choose to perform TESE followed by ICSI using fresh testicular spermatozoa (treatment TESE), spermatozoa was recovered in 19 patients (40%), with birth of 12 newborn. Of the 17 patients who choose to perform TESE followed by testicular sperm cryopreservation, spermatozoa were recovered in six patients (35%), with birth of one child. Of the patients who performed treatment TESE, nine went for a new cycle using cryopreserved spermatozoa. Of these, five patients had a previous failed treatment cycle (two patients, three newborn) and four with a previous success went for a new cycle (one patient, one newborn). Overall, the embryological and clinical rates were as follows: 52% of fertilization, 41% of blastocyst, 27% of implantation, 39% of live birth delivery and 47% of newborn. Of the 16 clinical pregnancies, 14 had a successful delivery (12 girls and 5 boys). The 17 newborns had a mean gestation time of 37.2 weeks (35.3% pre-term) and a mean newborn weight of 2781.3 g (37.5% low weight). Comparisons between cycles with fresh and frozen-thaw spermatozoa revealed higher fertilization and clinical pregnancy rates with fresh spermatozoa, with no differences regarding implantation or newborn rates. Of the 17 newborns, no abnormal karyotypes (n = 3) or numerical abnormalities in chromosomes 13, 18, 21, X and Y (n = 14) as evaluated by Multiplex Ligation-dependent Probe Amplification were observed. In conclusion, this study presents further data that reassures that men with KS have no increased risk of transmitting their genetic problem to the offspring.