Objective: Klinefelter syndrome (KS) represents a sex chromosome anomaly observed in approximately 1 in 500–600 phenotypic males. It is observed in 3% of infertile males and up to 11.9% of azoospermic males. KS manifests in either non-mosaic (47, XXY) or mosaic (47, XXY/46, XY) forms, with 85% of cases presenting as the non-mosaic 47, XXY karyotype. The average rate of surgical sperm retrieval in patients with KS is around 50%, ranging from 28% to 69%. In this study, we aimed to present the outcomes of microscopic testicular sperm extraction (micro-TESE) in patients with non-mosaic KS. Materials and Methods: The results of 61 patients diagnosed with KS, who presented to the Harran University Urology Clinic with azoospermia between 2017 and 2024, were retrospectively reviewed. Hormonal assessments, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol, and total testosterone (TT), were conducted for all patients, and their partners underwent gynecological evaluations for infertility. Testicular dimensions were recorded via scrotal ultrasonography. Patients were categorized into TESE-positive and TESE-negative groups, and parameters were compared between these groups. Results: The mean age of the patients was 29.0±5.1 years, and their mean infertility duration was 5.9±4.1 years. The sperm retrieval rate was 29.5% (n=18). Mean levels of FSH, LH, prolactin, estradiol, and TT were 44.9 IU/L, 23.3 IU/L, 10 nmol/L, 31.4 pmol/dL, and 219 ng/dL, respectively. Sperm was retrieved in 18 patients (29.5%), while no sperm was obtained in 43 (70.5%). No significant correlation was observed between patient age, testicular size, serum levels of FSH, LH, prolactin, estradiol, and TT, and sperm retrieval rates when comparing the TESE-positive and TESE-negative groups (P>0.005). Conclusion: In patients with non-mosaic KS, hormonal parameters, age, and infertility duration were not found to be significant predictors of the success of micro-TESE in sperm retrieval.