Abstract: Objective To investigate the clinical significance of chromosomal karyotype analysis of amniotic fluid in high-risk pregnant women in the second trimester. Methods Type-B ultrasound-guided amniocentesis was performed in 1 543 pregnant women with high-risk pregnancy in our hospital from January 2016 to December 2017 for cell culture, G banding and karyotype analysis. Results In 1 543 cases, 158 variant karyotypes were detected, with a variant rate of 10.24%, of which 101 were abnormal karyotypes, with an abnormal rate of 6.55%. There were 22 cases of Down syndrome, 13 cases of Edwards syndrome, 14 cases of sex chromosome, 2 cases of abnormal number of other chromosomes, 50 cases of abnormal structure and 57 cases of polymorphism changes, the detection rate was 1.43%, 0.84%, 0.91%, 0.13%, 3.24% and 3.69%, respectively. Conclusion High risk prenatal diagnosis combined with amniotic fluid chromosome analysis can effectively detect chromosomal abnormalities, which is important for preventing birth defects.