To evaluate the diagnostic efficiency of spent blastocyst culture medium (BCM) in noninvasive preimplantation genetic testing (niPGT) by comparing the karyotype concordance with corresponding inner cell mass (ICM) among initial trophectoderm (TE) biopsy, TE re-biopsy and BCM sampling. Re-analysis aneuploid/mosaic blastocysts donated for research by couples. A total of 26 blastocysts with aneuploid (n=23) or mosaic diagnosis (n=3) were donated for research from 12 couples with indications of preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR) or preimplantation genetic testing for aneuploidy (PGT-A), at Reproductive Medicine Research Center of the Sixth Affiliated Hospital of Sun Yat-Sen University, from July 2018 to July 2019. TE and ICM were biopsied (5-10 cells) in a routine procedure; TE was re-biopsied (more than 10 cells) at other sites of trophectoderm compartment. The MALBAC single-cell whole genome amplification (WGA) method was used to amplify DNA from all cells and BCM samples. Embryos were diagnosed as euploid, aneuploid or mosaic by NGS results. Embryos were classified as mosaic if the level of mosaicism ranged from 40% to 70%, while less than 40% was labeled as euploid and more than 70% as aneuploid. But for chr13, chr16, chr18 and chr21 chromosomes, the criterion for mosaicism was set to be 30%. The primary outcomes of this study, concordance rates with ICM biopsies, were compared among groups (initial TE biopsies, TE re-biopsies and BCM samples). The karyotype concordance of each two groups was defined as the presence of the same abnormal chromosome, especially in the target chromosome in different testing results compared. The clinical concordance of all four samples was defined as the concordance in embryo diagnosis, such as uniform aneuploid or not. To assess the diagnostic efficiency of BCM sample, false positive rate (FPR), false negative rate (FNR), sensitivity, specificity, accuracy, positive predictive value (PPV) and negative predictive value (NPV) were calculated according to the corresponding ICM results. Statistical analyses were performed with the Chi-Square test, Yates' correction, or Fisher's exact test accordingly when comparing frequencies or proportions. All statistical analysis was performed using SPSS 22.0. P<0.05 was considered as significant. For 23 embryos diagnosed as aneuploid by initial TE biopsy, 78.3% of initial TE samples, 87.0% of TE re-biopsies and 78.3% of BCM samples were concordant with corresponding ICM samples (P>0.05); but for 3 mosaic embryos, the concordance rates with ICM of these three groups were 0%, 100% and 100% (P<0.05), respectively. With the corresponding ICM result as the true result, sensitivity of both niPGT-A and initial TE were 100%; but the FPR of initial TE was higher than that of niPGT-A (100% vs. 0, P=0.100). NiPGT-A using spent BCM had similar diagnostic efficiency as TE-biopsy PGT-A. And in case of mosaic embryos, niPGT-A using BCM may be more reliable for predicting the karyotypes of ICM than initial TE biopsy.