Objective <p>This retrospective cytogenetic analysis aimed to characterize karyotypic profiles in a cohort of infertile Han Chinese couples undergoing assisted reproductive technology (ART) interventions.</p> Methods <p>G-banding karyotyping analysis was performed in 62,587 infertile couples from the outpatient department of the Institute of Women, Children and Reproductive Health, Shandong University (N-banding and C-banding technique or high-resolution technique if necessary) between January 2013 and December 2022.</p> Results <p>The overall prevalence of chromosomal abnormalities was 2.35% (2,938/125,174), with 2.04% (1,276/62,587) occurring in women and 2.66% (1,662/62,587) in men. Reciprocal translocation emerged as the predominant form of chromosomal abnormalities in the infertility population, followed closely by mosaicism. In the category of chromosomal polymorphisms, autosomal variations were identified in 8,372 cases, while sex chromosome variations were noted in 920 cases. The most frequently observed polymorphisms included 1qh+, 9qh+, 16qh+, and Yqh+ (add of the secondary constriction). The total incidence of chromosomal variations, including both abnormalities and polymorphisms, was significantly higher in males than in females, with a statistically significant difference (<i>P</i> &lt; 0.001).</p> Conclusions <p>Chromosomal variations are acknowledged as significant contributors to infertility, and infertile men possess an increased risk of chromosomal abnormalities or polymorphisms. Routine cytogenetic analysis, therefore, is strongly recommended for infertile couples undergoing ART for the most suitable treatment.</p>