A multiplex method for identifying the presence or absence of an aneuploidy of a target chromosome in a sample from a pregnant female subject, which comprises: (a) providing a plurality of amplification primer pairs, wherein each amplification primer pair specifically hybridizes with a nucleotide sequence species set, wherein: i) the nucleotide sequence species of a set are present on two or more different chromosomes, comprising a target chromosome and one or more reference chromosomes not associated with the aneuploidy; (ii) the nucleotide sequence species in a set differ by one or more mismatch nucleotides; and (iii) the nucleotide sequence species of a set are reproducibly amplified by a single pair of amplification primers relative to each other, wherein reproducibly amplified refers to consistency of amplification levels for the amplified nucleic acid species per unit template nucleic acid, wherein the template nucleic acid comprises a mixture of maternal nucleic acid and fetal nucleic acid; (b) contacting in one or more reaction vessels, under amplification conditions, extracellular nucleic acid of the sample comprising fetally derived and maternally derived nucleic acid with amplification primer pairs, wherein each reaction vessel comprises at least two amplification primer pairs and each amplification primer pair in a reaction vessel amplifies the nucleotide sequence species of a set, thereby producing a plurality of sets of amplified nucleic acid species; (c) determining the amount of each amplified nucleic acid species in each set by detecting the one or more mismatch nucleotides in each amplified nucleic acid species; (d) determining a ratio between the relative amount of (i) an amplified target nucleic acid species and (ii) an amplified reference nucleic acid species, for each set; and (e) identifying the presence or absence of an aneuploidy of a target chromosome based on the ratios from the plurality of sets of amplified nucleic acid species. (a) providing a plurality of amplification primer pairs, wherein each amplification primer pair specifically hybridizes with a nucleotide sequence species set, wherein: i) the nucleotide sequence species of a set are present on two or more different chromosomes, comprising a target chromosome and one or more reference chromosomes not associated with the aneuploidy; (ii) the nucleotide sequence species in a set differ by one or more mismatch nucleotides; and (iii) the nucleotide sequence species of a set are reproducibly amplified by a single pair of amplification primers relative to each other, wherein reproducibly amplified refers to consistency of amplification levels for the amplified nucleic acid species per unit template nucleic acid, wherein the template nucleic acid comprises a mixture of maternal nucleic acid and fetal nucleic acid; (b) contacting in one or more reaction vessels, under amplification conditions, extracellular nucleic acid of the sample comprising fetally derived and maternally derived nucleic acid with amplification primer pairs, wherein each reaction vessel comprises at least two amplification primer pairs and each amplification primer pair in a reaction vessel amplifies the nucleotide sequence species of a set, thereby producing a plurality of sets of amplified nucleic acid species; (c) determining the amount of each amplified nucleic acid species in each set by detecting the one or more mismatch nucleotides in each amplified nucleic acid species; (d) determining a ratio between the relative amount of (i) an amplified target nucleic acid species and (ii) an amplified reference nucleic acid species, for each set; and (e) identifying the presence or absence of an aneuploidy of a target chromosome based on the ratios from the plurality of sets of amplified nucleic acid species.