BACKGROUND The accurate assignment of alleles embedded within trisomic or duplicated regions is an essential prerequisite for assessing the combined effects of single-nucleotide polymorphisms (SNPs) and genomic copy number. Such an integrated analysis is challenging because heterozygotes for such a SNP may be one of 2 genotypes—AAB or ABB. Established methods for SNP genotyping, however, can have difficulty discriminating between the 2 heterozygous trisomic genotypes. We developed a method for assigning heterozygous trisomic genotypes that uses the ratio of the height of the 2 allele peaks obtained by mass spectrometry after a single-base extension assay. METHODS Eighteen COL6A2 (collagen, type VI, alpha 2) SNPs were analyzed in euploid and trisomic individuals by means of a multiplexed single-base extension assay that generated allele-specific oligonucleotides of differing Mr values for detection by MALDI-TOF mass spectrometry. Reference data (mean and SD) for the allele peak height ratios were determined from heterozygous euploid samples. The heterozygous trisomic genotypes were assigned by calculating the z score for each trisomic allele peak height ratio and by considering the sign (+/−) of the z score. RESULTS Heterozygous trisomic genotypes were assigned in 96.1% (range, 89.9%–100%) of the samples for each SNP analyzed. The genotypes obtained were reproduced in 95 (97.5%) of 97 loci retested in a second assay. Subsequently, the origin of nondisjunction was determined in 108 (82%) of 132 family trios with a Down syndrome child. CONCLUSIONS This approach enabled reliable genotyping of heterozygous trisomic samples and the determination of the origin of nondisjunction in Down syndrome family trios.