Objective To analyse karyotype of chromosome in patients with infertility and sperm donors, and explore its clinical significance. Methods A total of 967 infertile patients (infertility group) and 3 184 semen donors (donor group) were selected for analysis of karyotype of chromosome, and the incidence and types of abnormal karyotypes were compared between two groups. Results The incidence of abnormal karyotypes in infertility group was significantly higher than that in donor group (14.06% vs 3.39%, P<0.01). The main type of abnormal karyotype in infertility group was non-polymorphic chromosomal abnormalities, whose incidence was significantly higher than that in donor group (9.62% vs 0.25%, P<0.05). The main type of abnormal karyotype in donor group was polymorphic chromosomal abnormalities, whose incidence was not significantly different from that in infertility group (3.14% vs 4.44%, P>0.05). Besides, two abnormal karyotypes [46,XY,t(3;12)(p23;q24) and 46,XY,inv(20)(p13;q13.1)] were first reported worldwide in infertility group. Conclusion Chromosome non-polymorphism abnormalities is one of the major causes for male infertility, and karyotype analysis for infertile patients and sperm donors is of great significance to reduce birth defects.