A chromosome number determination method of chromosomes of interest, comprising: a step of performing multiplex PCR for simultaneously amplifying a plurality of loci on the chromosomes using genomic DNA extracted from a single cell or a small number of cells as templates, wherein the number of loci on the chromosomes of interest is greater than or equal to 80 per chromosome, wherein a plurality of primer sets used in the multiplex PCR are designed through a method for designing primer sets used in the polymerase chain reaction, the method for designing primer sets including a target locus selection step of selecting a target locus for designing primer sets used in the multiplex PCR from the plurality of loci, a primer candidate base sequence generation step of generating at least one base sequence of a primer candidate for amplifying the target locus regarding each of a forward-side primer and a reverse-side primer based on a base sequence in a vicinity region of the target locus on the chromosomes, a local alignment step of obtaining a local alignment score by performing pairwise local alignment on the base sequence of the primer candidate for amplifying the target locus under a condition that a partial sequence to be subjected to comparison includes the 3' terminal of the base sequence of the primer candidate for amplifying the target locus, a first stage selection step of performing first stage selection of the base sequence of the primer candidate for amplifying the target locus based on the local alignment score obtained in the local alignment step, a global alignment step of obtaining a global alignment score by performing pairwise global alignment on a base sequence which has a sequence length of three bases and includes the 3' terminal of the base sequence of the primer candidate for amplifying the target locus, a second stage selection step of performing second stage selection of the base sequence of the primer candidate for amplifying the target locus based on the global alignment score obtained in the global alignment step, and a primer employment step of employing the base sequence of the primer candidate which has been selected in both of the first stage selection step and the second stage selection step as the base sequence of the primer for amplifying the target locus, and wherein both steps of the local alignment step and the first stage selection step are performed before or after both steps of the global alignment step and the second stage selection step, or performed in parallel with both steps of the global alignment step and the second stage selection step. a step of performing multiplex PCR for simultaneously amplifying a plurality of loci on the chromosomes using genomic DNA extracted from a single cell or a small number of cells as templates, wherein the number of loci on the chromosomes of interest is greater than or equal to 80 per chromosome, wherein a plurality of primer sets used in the multiplex PCR are designed through a method for designing primer sets used in the polymerase chain reaction, the method for designing primer sets including a target locus selection step of selecting a target locus for designing primer sets used in the multiplex PCR from the plurality of loci, a primer candidate base sequence generation step of generating at least one base sequence of a primer candidate for amplifying the target locus regarding each of a forward-side primer and a reverse-side primer based on a base sequence in a vicinity region of the target locus on the chromosomes, a local alignment step of obtaining a local alignment score by performing pairwise local alignment on the base sequence of the primer candidate for amplifying the target locus under a condition that a partial sequence to be subjected to comparison includes the 3' terminal of the base sequence of the primer candidate for amplifying the target locus, a first stage selection step of performing first stage selection of the base sequence of the primer candidate for amplifying the target locus based on the local alignment score obtained in the local alignment step, a global alignment step of obtaining a global alignment score by performing pairwise global alignment on a base sequence which has a sequence length of three bases and includes the 3' terminal of the base sequence of the primer candidate for amplifying the target locus, a second stage selection step of performing second stage selection of the base sequence of the primer candidate for amplifying the target locus based on the global alignment score obtained in the global alignment step, and a primer employment step of employing the base sequence of the primer candidate which has been selected in both of the first stage selection step and the second stage selection step as the base sequence of the primer for amplifying the target locus, and wherein both steps of the local alignment step and the first stage selection step are performed before or after both steps of the global alignment step and the second stage selection step, or performed in parallel with both steps of the global alignment step and the second stage selection step.