Purpose <p>To evaluate the association between the method of sperm retrieval (ejaculated versus testicular sperm) and embryonic mosaicism in infertile couples with severe male factor infertility undergoing in vitro fertilization (IVF) with preimplantation genetic testing for aneuploidy (PGT-A).</p> Methods <p>This retrospective study analyzed 298 patients with severe male factor infertility undergoing IVF with PGT-A from 2019 to 2025. Patients were divided into two groups based on sperm source: ejaculated sperm (<i>n</i> = 229) and testicular sperm obtained via testicular sperm extraction (TESE) (<i>n</i> = 69). All cycles used fresh sperm collected on the day of oocyte retrieval. The primary outcome was the rate of embryonic mosaicism among biopsied blastocysts. Secondary outcomes included euploidy, aneuploidy, mosaicism level, inconclusive embryo count, clinical pregnancy and live birth rates.</p> Results <p>A total of 1124 embryos were biopsied (250 from testicular sperm extraction; 874 from ejaculate). Mosaicism rates were similar between testicular and ejaculated sperm groups (9.6% vs. 9.4%, <i>p</i> = 0.62). Sperm retrieval method was not associated with mosaicism (OR 0.98, CI 0.61–1.58), euploidy (OR 0.90, CI 0.70–1.08), or aneuploidy (OR 0.89, CI 0.60–1.06). High- and low-level mosaicism were also comparable between groups (OR 0.96, CI 0.75–1.18 and OR 0.99, CI 0.81–1.22). No differences were observed in clinical pregnancy and live birth rates among cohorts.</p> Conclusions <p>Sperm retrieval technique does not influence embryonic mosaicism rates in patients with severe male factor infertility. These findings support patient counseling by indicating that invasive procedures should not be pursued solely for the purpose of reducing mosaicism rates.</p>