Many ticks of the genus Amblyomma are vectors of human pathogens, and the correct species identification is medically and epidemiologically important. Morphological identification is time-consuming and requires a high level of expertise. Identification of engorged, immature, or damaged ticks and the differentiation of closely related species remain problematic. Here, we report the development of a real-time TaqMan assay for the genomic identification and differentiation of Amblyomma americanum (L.), Amblyomma cajennense (F.), and Amblyomma maculatum (Koch), which are human-biting species found in the eastern United States. New species-specific sets of oligonucleotides for the multiplex reaction that detect and differentiate the ITS2 genomic regions of three target species were designed using Visual OMP; the previously published A. americanum oligonucleotide set was also incorporated into our assay. Specificity and sensitivity tests for two multiplex master mixes using different A. americanum sets were performed using individual and pooled samples of adult, nymphal, and larval ticks, and optimization procedures were applied. The multiplex assay successfully differentiates between genomes of three target species and does not cross-react with DNAs of ticks from other genera. Rare cases of nonspecific amplification occurred with DNAs of A. imitator and Amblyomma triste Koch misidentified as A. americanum and A. maculatum, respectively. However, this cross-reaction does not diminish the usefulness of the developed assay east of the 95th meridian, where neither A. imitator nor A. triste are found. Two master mixes incorporating the previously published or newly developed A. americanum sets are being recommended for identification of individual ticks or pooled samples, respectively.