Abstract: Objective To evaluate the effects combining in situ amniotic fluid culture with chromosomal microarray analysis ( CMA) in prenatal diagnosis of fetal chromosomal abnormalties. Methods A retrospective analysis was conducted on 3 133 pregnant women who underwent amniocentesis at Huaihua Maternal and Child Health Hospital from October 2018 to February 2023 due to different indications for prenatal diagnosis. The detection rates and differences of in situ amniotic fluid culture and CMA in detecting fetal chromosomal abnormalities were compared. Results Among the 3 133 samples, the detection rates increased by 0.95% ( in situ culture) and 2.36% ( CMA) in the double-indication group compared with the single-indication group. The combined testing detected 796 abnormal cases ( 25 .41%)，including 300 cases (9.58%) by in situ culture and 706 cases ( 22.53%) by CMA. Both techniques detected 145 aneuploidies and 31 large segmental structural abnormalities ，while CMA aditionally detected 169 copy number variants (CNV)，loss of heterozygosity ( LOH)，absence of heterozygosity ( AOH) / region of homozygosity ( ROH) and uniparental disomy (UPD), and in situ culture additionally detected 11 balanced rearrangements. They jointly detected 23 cases of mosaicism (0.73%). Conclusion In situ amniotic fluid culture and CMA complement each other. Their combined application can significantly improve the detection rate of fetal chromosomal abnormalities and provide more detailed and accurate information for prenatal genetic counseling , thus facilitating pregnant women to decide continuing or terminating the pregnancy.