Although interspecific chimeras can be identified using laborious techniques, more accurate and rapid detectable methods need to be established. The present study develops a nested polymerase chain reaction (nPCR) assay to detect human-mouse chimeras. A set of previously validated outer primers, specific to the human SRY gene, was used for conventional one-step PCR, while the inner primers for nPCR were designed. The specificities of PCR and nPCR assays were examined; both primer sets yielded PCR amplification products from male human epidermis-derived mesenchymal stem cell-like pluripotent cell DNA but no amplification products from negative control DNA. The sensitivity of this nPCR was determined using mixed DNA. Measurable amplification of SRY transcripts was a male human to female mouse DNA ratio of 1:10,000. We then tested the nPCR assay on tissues from female mouse chimeras. The nPCR products were selected randomly for sequencing and positive samples were further analyzed by fluorescence in situ hybridization (FISH) using specific probes for the human SRY gene and by immunofluorescence staining for species-specific markers. There was 100% concordance among nPCR, FISH, and immunofluorescence results, and the nPCR product sequences were consistent with the human SRY gene. Taken together, we developed a new, highly specific, sensitive, and reliable method to detect human-mouse chimeras.